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- Publisher
- Wiley
- Copyright
- Copyright © 2012 John Wiley & Sons, Ltd.
- ISSN
- 1522-7235
- eISSN
- 1522-7243
- DOI
- 10.1002/bio.2341
- pmid
- 22505334
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Abstract
Preliminary attempt for pharmacometrics assay by bioluminescence imaging in Drosophila embryogenesis Ryutaro Akiyoshi and Hirobumi Suzuki Research & Development Division, Olympus Corporation E‐mail: ryutaro_akiyoshi@ot.olympus.co.jp Drosophila has been widely used as a model animal for genetic, developmental and physiological studies due to easy crossing and breeding and short life cycle. Nowadays, it could be used for pharmacometirc assay related to human disease. Because biologically important inventions of morphogenesis have been done in Drosophila, and it is confirmed that developmental pathways in morphogenesis are conserved through evolution. For example, wingless and armadillo genes related to segment polarity forming signal system in Drosophila are homologous with Wnt and beta‐catenin genes in mammals, and some factors of the Wnt/beta‐catenin signaling pathway relate to not only morphogenesis but also cell growth, transformation and so on. Therefore, Drosophila could be an alternative model of mammals for pharmacometrics assay. In order to develop the pharmacometrics assay system of drug‐induced effect during embryogenesis, we established bioluminescent transgenic Drosophila of the armadillo and applied it to whole Drosophila bioluminescence imaging. We cloned armadillo (arm) promoter region from D. melanogaster genome and constructed a bioluminescent reporter vector of arm::ELuc by inserting the arm promoter and click beetle luciferase gene, (Emerald Luc, Toyobo), into KP124 vector. The arm::ELuc vector was injected into fertilized eggs of D. melanogaster by microinjection method, and we established transgenic D. melanogaster strain undergoing processes of crossing with another strain. The arm gene expression pattern of this strain until hatching was observed by the bioluminescence microscope (LV200, Olympus) together with morphogenetic movements for 18 hours. The arm activity through blastula stage was observed in blastoderms slightly, and increased in whole embryo at the beginning of gastrulation, and then it suddenly decreased at the end of gastrulation. In the process of midgut formation going with germ band shortening in parallel, the arm promoter activity increased in midgut primodium dramatically and continued until hatching. On the other hand by ionomycin treatment that inhibits Wnt signal, the arm promoter activity was depressed until the end of gastrulation and increased at the timing for midgut formation. In spite of this unusual gene expression by ionomycin treatment, the embryo hatched normally. These results suggest that bioluminescence imaging of promoter assay in Drosophila embryogenesis could provide a new pharmacometrics method for evaluating not only gene expression pattern influenced by drugs but also morphological changes during the developmental process. Effect of tritium on bioluminescent systems MA Alexandrovaa, GA Badunb and NS Kudryashevaa,c aSiberian Federal University, Svobodniy 79, 660041, Krasnoyarsk, Russia bMoscow State University, Chemistry Dept., 119991, Moscow, Russia cInstitute of Biophysics SB RAS, Akademgorodok 50, 660036. Krasnoyarsk, Russia Bacterial bioluminescence (BL) assay has been used to monitor toxicity of radionuclide solutions for the first time in (1,2); toxicity of alpha‐radionuclides Am‐241 and U‐(235+238) was studied using bioassays based on Photobacterium phosporeum. Details of BL kinetics and role of peroxides in activation and inhibition of BL intensity in Am‐241 solutions were discussed in (3). Tritium, beta‐emitting nuclide, is one of the most widespread radionuclides, its content increases in environment now. Chronic effect of tritium (0.25–10 MB q/L) on growth and bioluminescence of the bacteria was studied in (4). Purpose of the current study was to compare effects of tritiated water (0.0002–100 MBq/L) on different BL systems. Three BL assay systems were applied: (1) intact luminous bacteria P. phosphoreum 1883 IBSO; (2) a microbiotest 677F preparation i.e. lyophilized luminous bacteria cells of P. phosphoreum 1883 IBSO; and (3) a kit of reagents for analytical BL, which included lyophilized preparations of luciferase (0.5 mg/mL) and NADH:FMN‐oxidoreductase (0.15 units of activity). All assay systems were produced at the Institute of Biophysics SB RAS, Krasnoyarsk, Russia. Tritiated water (HTO, “Isotop”, Russia) was used as a source of beta‐radiation. HTO was added to BL solutions. Radioactivity of the solutions was: 0.0002, 0.002, 0.02, 0.2, 2.0, 10, 20, 50, 100 MBq/L. The samples were kept at +4 °C, their BL intensity was measured by ‘TriStar Multimode Microplate Reader LB 941’ (Berthold Technologies). BL kinetic curves in the presence of HTO were compared for three BL systems. Activation of BL of enzymatic system (3) was observed at lower radioactivity of tritium solutions (A<10 MBq/L) and BL inhibition – at higher radioactivity (A>10 MBq/L). Effects of tritium on bacterial systems (1–2) were of different type: BL activation was observed at initial time of exposure to tritium (less than 50 h for system (1), and 30 h for system (2)), and BL inhibition ‐ at final time of exposure. The results show that the resistance of the BL function to tritium increases from enzymes to cells, i.e. with increase of complexity of the systems. It was assumed that BL activation and inhibition by tritium can be related to intensification of the processes of electron density redistribution in the course of beta‐decay. BL activation is discussed in terms of “radiation hormesis”. Acknowledgements The work was supported by Grants from RFBR N09‐08‐98002‐Sibir_a; RFBR N10‐05‐01059‐a Ministry of Education RF N2.2.2.2/5309, ‘Leading Scientific School’ N 1211.2008.4; Program ‘Molecular & Cellular Biology’ of RAS. References Rozhko TV, Kudryasheva NS. Effect of low‐level α‐radiation on bioluminescent assay systems of various complexity. Photochem. Photobiol. Sci. 2007;6:67–70. Rozhko T, Kudryasheva N, Aleksandrova M, et al. Comparison of Effects of Uranium and Americium on Bioluminescent Bacteria. J. Siberian Federal Univ, Biology, 2008;1:60–4. M Alexandrova, T Rozhko, et al. Effect of americium‐241 on luminous bacteria. Role of peroxides. J. Environ. Radioactiv. 2011;102:407–11. Alexandrova MA, Rozhko TV, et al. Effect of tritium on growth and bioluminescence of bacteria P.Phosphoreum. Radiat. Biol. Radioekol. 2010;6:613–8. Thermoinactivated photoprotein obelin: fluorescence peculiarities RR Alievaa, NV Belogurovab, AS Petrovaa and NS Kudryashevaa,b aSiberian Federal University, Krasnoyarsk, 660041, Russia; E‐mail: alieva_rosa@mail.ru bInstitute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia; E‐mail: n_qdr@yahoo.com Discharged obelin, being a fluorescent protein, is a perspective fluorescence marker for biological and medical investigations. Its fluorescence spectra are complex, their components correspond to different fluorescent species – different forms of coelenteramide (1). Variation of calcium ion concentration is known to change fluorescence spectra of Ca2+‐discharged obelin significantly (2). The discharged obelin can be obtained in two ways: by addition of Ca2+ (Ca2+‐discharged obelin) and under exposure to higher temperature (thermo‐discharged obelin). In the current study, we obtained the thermo‐discharged obelin by 3‐h exposure to 40 °C, analyzed its spectra and compared to those of the Ca2+‐discharged obelin. Complex fluorescence spectra were deconvolved into components using Gauss‐based distribution and method of second derivative. One spectral component was found in excitation spectrum, and two components – in emission spectrum of the thermo‐discharged obelin. Three components were found in the excitation spectrum of Ca2+‐discharged obelin and four components – in its emission spectrum (2). Contributions of the components to experimental spectra were calculated. Maximal contribution of 410‐nm‐component into emission spectrum of thermo‐discharged obelin was found, while the spectrum of Ca2+‐discharged obelin was characterized by the maximal contribution of 510‐nm‐component (1,2). According to the previous study (3), the 410‐nm‐component can be assigned to protonated coelenteramide species, while the 510 nm‐component – to the deprotonated one. Our data suggest that proton‐transfer process in excited coelentaramide is less effective in thermo‐discharged obelin as compared to Ca2+‐discharged one. Hence, in our study, the difference in fluorescence spectra of thermo‐discharged and Ca2+‐discharged obelin was found and described. References Belogurova NV, Kudryasheva NS, Alieva RR, Sizykh AG. Spectral components of bioluminescence of aequorin and obelin. J. Photochem. Photobiol. B. 2008;92:117–22. Belogurova NV, Kudryasheva NS. Discharged photoprotein obelin: fluorescence peculiarities. J. Photochem. Photobiol. B. 2010;101:103–8. 3. Shimomura O, Teranishi K. Light‐emitters involved in the luminescence of coelenterazine. J. Luminescence. 2000;15:51–8. Molecular phylogeny of the neotropical bioluminescent beetles DT Amarala,b,c*, FGC Arnoldid and V Viviania,b,c aLaboratory of Biochemistry and Biotechnology of Bioluminescence bDepartment of Evolutional Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil cGraduate Program of Biotechnology and Environment Monitoring, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Sorocaba, SP, Brazil dRibeirão Preto School of Medicine, São Paulo University (USP), Ribeirão Preto, São Paulo, Brazil E‐mail: danilo.trabuco@gmail.com Bioluminescence in Coleoptera is found mainly in the Elateroidea superfamily, which includes several families of non‐luminescent beetles, with bioluminescent species occurring, in Lampyridae (fireflies), Phengodidae (railroadworms) and Elateridae (click‐beetles). Despite using the same bioluminescent system, involving a benzothiazolic luciferin, ATP and homologous luciferases, the phylognetic relationships of these families is still partially unclear, and it is unclear whether bioluminescence in these families share a common origin or may have evolved independently. To better understand the phylogeny of bioluminescent Elateroidea and the origin of bioluminescence in this group, we sequenced and analyzed the mitochondrial gene NADH2, the 28S rDNA and the sequences of cloned luciferases of different Brazilian taxa, using Maximum‐parsimony, Neighbor‐Joining and Bayesian methods. The high support values for the branches, obtained in these analyses, indicate the monophyly of Elateridae, Phengodidae, Lampyridae, Lycidae and Cantharidae and the relationship of these families. The families Elateridae and Phengodidae formed a sister‐group, as well as Cantharidae and Lampyridae, with high values of support when we used the mitochondrial and nuclear genes, in contrast with former phylogenetic analysis based on the primary sequences of cloned luciferases that clustered Phengodidae and Lampyridae. We examined seven elaterid species, five of them being luminescent. Our data showed two distinct clusters, the first one represented by Pyrophorus, Hapsodrilus, Fulgeochlizus and Pyrearinus genera and the second represent by Agrypnus and Conoderus genera, corresponding to luminescent and non‐luminescent groups, respectively. The first one seems to be more derived than the second cluster, supporting the hypothesis that the ancestor of this group was non‐luminescent. The Lampyridae family formed four clusters, representing the subfamilies Lampyrinae, Photurinae, Amydetinae and Luciolinae. Our data also supports the classification of Amydetes spp as an independent subfamily, Amydetinae. Within Phengodidae, we observed the monophyly of the subfamily Rhagophtalminae and Phengodinae. Altogether, these results support the hypothesis that bioluminescence may have originated independently in these three families. Financial support: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo; n° 2010/02868‐0) and CNPq, Brazil. A New luciferase from Fulgeochlizus bruchi, a Brazilian click‐beetle with a single abdominal lantern: cDNA cloning, molecular properties and evolution (Coleoptera:Elateridae) DT Amarala,b,c*, RA Pradoa,b,c and V Viviania,b,c aLaboratory of Biochemistry and Biotechnology of Bioluminescence bDepartment of Evolutional Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil cGraduate Program of Biotechnology and Environment Monitoring, Federal University of São Carlos (UFSCar), Campus of Sorocaba, Sorocaba, SP, Brazil E‐mail: danilo.trabuco@gmail.com Bioluminescent click‐beetles (Elateridae) produce one of the widest range of colors (λMax = 534–594 nm) among bioluminescent beetles from thoracic and abdominal lanterns. The Brazilian Fulgeochlizus bruchi click‐beetle, displays only a functional abdominal lantern which produces a bright green bioluminescence which is used for courtship, missing functional thoracic lanterns. The cDNA for this luciferase was cloned, showing higher identity with the dorsal luciferases of Pyrophorus genus; 50% identity with the luciferases from railroad worms and 48% with luciferase from fireflies. These results suggest that the pH‐insensitive click beetle and railroad worm luciferases could be closer than previously shown, suggesting the remote possibility of sharing a common ancestor. This luciferase displays one of the most blue‐shifted spectra (λmax = 540 nm) among click beetle luciferases, which is pH‐insensitive from pH 7,5 to 9,5; a slow decay rate (KD = 0,0046) and a low KM (KM = 12,3 μM) for luciferin when compared to other studied click‐beetles luciferases, and higher optimum pH in relation to other beetle luciferases. Comparison of the primary structures of click beetle luciferases showed that several residues in the region 220–360 are conserved in green and yellow‐green emitting luciferases, being substituted among yellow and orange emitting luciferases. Financial support: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo; n° 2010/02868‐0) and CNPq (Conselho Nacional de Pesquisa), Brazil. Peroxyoxalate chemiluminescence in aqueous medium: concurrence between hydrolysis and perhydrolysis Felipe A. Augusto*, Fernando H. Bartoloni and Wilhelm J. Baader Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508‐000, São Paulo, SP, Brazil E‐mail: felipe.augusto@usp.br The peroxyoxalate system is used as analytical tool to detect different types of substances, with the typical advantages that chemiluminescence methods usually provide: simplicity, low costs and good detection limits (1). However, as many of the analytical applications require the use of aqueous medium and only little information concerning the mechanism of this reaction in this medium has been obtained (2), mechanistic studies were performed in order to elucidate the influence of water on the kinetic behavior of this system and to understand the role of water in this reaction. The peroxyoxalate reaction was performed using bis(2,4‐dinitrophenyl) oxalate (DNPO) and hydrogen peroxide, with imidazole (IMI‐H) as catalyst and 2,5‐diphenyloxazole as activator. Solvent mixtures containing 0, 10, 30 and 50% of water in dimethoxyethane were utilized in order to evaluate reactivity and medium effects. Attempting to obtain more information about the system, the direct reaction between DNPO and hydrogen peroxide was also investigated. In order to utilize water as a reagent, no reaction medium, studies were performed using water concentrations comparable to those of the other reagents. Observed rate constants (kobs) were determined from the kinetic emission intensity decay curves and the dependence of these rate constants with the concentrations of imidazole and hydrogen peroxide was measured for each solvent mixture utilized. From the slope of the linear correlations between the rate constants and the reagent concentrations, the bimolecular rate constants (kIMI‐H and kH2O2) were determined. From the intercept of the correlation between kobs and (H2O2), it was possible to obtain the hydrolysis rate constant (kh) and the intercept of the correlation between kobs and (IMI‐H) allows the determination of the rate constants for the uncatalyzed reaction (kNper and kNhyd). The combination of uni‐ and bimolecular rate constants determined allows the calculation of termolecular rate constants for the catalyzed perhydrolysis (kCper) and catalyzed hydrolysis (kChyd) reactions. These experimental results allowed us to formulate a mechanistic scheme for the peroxyoxalate reaction in these conditions, with concurrent pathways of imidazole catalyzed and uncatalyzed perhydrolysis and hydrolysis reactions. The values calculated for the hydrolysis and perhydrolysis rate constants (kNhyd = 2,5 10–2 L mol–1 s–1, kNper = 0,5 L mol–1 s–1, kChyd = 20 L2 mol–2 s–2 and kCper = 2,7 104 L2 mol–2 s–2) show that the imidazole catalyzed hydrolysis is three orders of magnitude more efficient than the hydrolysis, whereas the neutral perhydrolysis is only 20 times more efficient. These results indicate that the peroxyoxalate reaction can be utilized in these conditions for analytical applications in aqueous medium. References Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MM de M, Santos APF, Santos CS, Oliveira S, Augusto FA, Pagano APE, Baader WJ. Quim. Nova, 2011;34:544. Baader WJ, Stevani CV, Bastos EL. In: The Chemistry of Peroxides, Rappoport Z, ed., John Wiley & Sons, Chichester, 2006, vol. 2, cap. 16, p. 1211. Heterogeneous binding of 1‐anilinonaphtalene‐8‐sulfonate to bacterial luceferase from steady‐state and time‐resolved fluorescence TI Avsievicha, EV Nemtsevaa,b, MA Gerasimovaa and VA Kratasyuka,b aDep. of Biophysics, Siberian Federal University, 79 Svobodny Pr., Krasnoyarsk, 660041, Russia bLab. of Photobiology, Institute of Biophysics SB Russian Academy of Science, 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia To study the binding properties of the proteins a fluorometric titration by 8‐anilinonaphthalene‐1‐sulfonic acid (ANS) is widely used. This probe is popular because its fluorescence is dramatically enhanced after binding to proteins. Recently it was shown that the properties of external and internal binding sites of the macromolecules can be defined from fluorescence lifetimes distribution of ANS (1). To characterize the binding sites of bacterial luciferase we studied the interaction of ANS with this protein from steady‐state and time‐resolved fluorescence of the probe. Bacterial luciferase is a heterodimeric enzyme with molecular mass of about 80 kDa that catalyzes the bioluminescence reaction and gives basis for many analytical tools (2). The exact number and affinities of its binding sites have not been determined yet. In this work ANS fluorescence (1–70 μM) was studied at a fixed concentration of Photobacterium leiognathi luciferase (8 μM) in phosphate buffer solution (0.05 M, pH 6.9). Steady‐state fluorescence spectra and time‐resolved fluorescence decay were measured using spectrofluorimeter Fluorolog 3–22 (Horiba Jobin Yvon, France) equipped with TCSPC. Absorption spectra were recorded with spectrophotometer Lambda 35 (Perkin Elmer, USA). All data were examined for distortion from scattering and inner filter effect and were corrected if necessary. A typical hyperbolic response from ANS emission at 470 nm to increasing concentration was obtained (fig. 1). The standard Scatchard analysis of the titration data was conducted. The concentrations of the bound probe were estimated using proportionality coefficient between fluorescence intensity and ANS concentration calculated from the linear part of the hyperbolic curve. The downward‐curved Scatchard plot was obtained indicating either cooperative effect or two different kind of binding sites on the same macromolecule (3). Assuming the second reason the parameters of asymptotes were used to calculate dissociation constants Kdi and number of binding sites ni: Kd1=1.0 ± 0.2 μM, Kd2=5.7 ± 0.3 μM, n1=2.2 ± 0.6 and n2=3.2 ± 0.2. Alternatively the ANS fluorescence decays were measured at the wavelengths from 420 to 615 nm with increment of 5 nm (for mixture of 100 μM of ANS and 8 μM of protein). The global analysis of decays (4) revealed two types of emitters with short and long lifetimes (τ1=7.6 ns and τ2=17.7 ns), those could be attributed to the external and internal binding sites of luciferase macromolecule (1). The fractional intensities of each component were calculated for the data set with different ANS concentrations obtained during titration. It allowed to estimate the contribution of every type of emitter into steady‐state fluorescence intensity and to decompose the titration curve into two individual species (fig. 1). Nonlinear fit of separated curves according formula from (1) defined the following binding parameters: Kd1=24.9±15.9 μM, Kd2=4.3±0.8 μM, n1=3.7±1.2 and n2=1.5±0.1. Figure 1. Deconvolution of ANS fluorescence titration curve into lifetime components (markers) and their nonlinear fitting (straight lines). Thus, accordance of the means from the two approaches shows that steady‐state data analysis could indicate the heterogeneity of fluorescent probe binding, but it is insufficient to characterize separated species of emitters. The time‐resolved experiments are necessary to obtain the reliable information about affinities and numbers of biding sites. References Gasymov OK, Abduragimov AR, Glasgow BJ. Arch. Biochem. Biophys. 2007;468:15. Roda A, Pasini P, Mirasoli M, Michelini E, Guardigli M. Trends in biotechnology 2004;22(6):295. Bordbar AK, Saboury AA, Moosavi‐Movahedi AA. Biochem Education 1996;24(3):172. van Stokkum IHM, Larsen DS and van Grondelle R. Biochim. Biophys. Acta. 2004;1657(2–3):82. Peroxyoxalate chemiluminescence: mechanisms and applications Wilhelm J. Baader*, Felipe A. Augusto, Fernando H. Bartoloni and Luiz F. M. L. Ciscato Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508‐000 São Paulo, SP, Brazil *E‐mail: wjbaader@iq.usp.br In this contribution, a general revision about the peroxyoxalate chemiluminescence will be given with special attention to mechanistic aspects of this highly efficient chemiluminescent transformation; however, we will also refer to the general principles of analytical applications of the system. Even almost fifty year after the discovery of the peroxyoxalate reaction by E. A. Chandross (1) in the early nineteen sixties, the mechanism of this complex transformation, containing innumerous possible consecutive and parallel reaction steps, (2) is still not yet well understood, specifically with respect to the nature of the high‐energy intermediate structure and the exact mechanism of the chemiexcitation step, where this high‐energy intermediate interacts with an appropriate activator, leading to electronically excited state formation (3). Chemiluminescence reactions may be divided in three main parts: (i) formation of a high‐energy intermediate, in one or more chemical reaction steps, all on the ground state energy surface; (ii) decomposition of the high‐energy intermediate, eventually with the participation of another reagent, leading to electronically excited state formation, in the so‐called chemiexcitation step; (iii) decay of this excited state to the ground state accompanied by fluorescence or phosphorescence emission, depending on the multiplicity of the excited state. Following the fundamental work by Rauhut's group (2), the initial steps of the peroxyoxalate reaction have been subject to several kinetic studies using different oxalic esters and analogous derivatives, base and nucleophilic catalysts, a wide variety of chemiluminescent activators as well as many different reaction conditions. Imidazole has been widely employed as catalyst leading to reproducible reaction conditions and it was shown that this compound acts as basic as well as nucleophilic catalyst (4). The exact structure of the high‐energy intermediate is still a matter of discussion in the literature; in order to contribute to this controversy we have studied the chemiluminescence properties of peroxalic acids derivatives and obtained evidence on the occurrence of 1,2‐dioxetanedione as the high‐energy intermediate (5). The chemiexcitation step, where the high‐energy intermediate interacts with an appropriate activator, has been shown to involve a rate‐limiting electron or charge transfer, and this step has been directly observed by kinetic measurements in certain reaction conditions (6). In the final part of this contribution the principle of analytical applications of peroxyoxalate chemiluminescence will be discussed and some recent representative examples be given. References Chandross EA. Tetrahedron Lett. 1963;12:761. Rauhut MM. Acc. Chem. Res. 1969;2:80. Bartoloni FH, Bastos EL, Ciscato LFML., Peixoto MM de M, Santos APF, Santos CS, Oliveira S, Augusto FA, Pagano APE, Baader WJ. Quim. Nova, 2011;34:544. Da Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 1996;2:1645. Ciscato LFML, Bartoloni FH, Bastos EL, Baader WJ. J. Org. Chem. 2009;74:8974. Diffraction of the bioluminescent light of the firefly Anurup Gohain Barua Department of Physics, Gauhati University, Guwahati‐781014, India E‐mail: agohainbarua@yahoo.com A Hilger Analytical grating of lines per inch of 15000 has been used to study diffraction of the light from specimens of the Indian species of the firefly, Luciola praeusta Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae), along with two Japanese species Luciola cruciata and Luciola lateralis. The diffraction patterns are quite similar. One of those, from a specimen of Luciola praeusta is shown in Figure 1(a) along with the intensity profile in Figure 1(b). The central principal maximum is yellow. It is worth mentioning here that for a polychromatic source, the central maximum is of the same colour as the source itself. In other orders of maxima, different colours appear approximately as per the grating equation(a + b)sin θ = nλ, where a + b is the grating element, θ is the angle of diffraction, n = 0, 1, 2, … and λ is the wavelength of light. The striking feature of the pattern is that the yellow colour, which is so predominant in the central principal maximum, gets suppressed by green and red colours in first and second order maxima. In the second order maximum, this band becomes so narrow that it is visible only with some difficulty only. The pattern formed by the grating when there is no separation between it and the source is shown in Figure 2. That is, the grating is placed right on top of the firefly positioned upside down in cotton wool. Different orders of principal maxima gradually emerge and move away from this when the grating is slowly moved away from the lantern. Yellow rings are clearly noticeable in the figure. Figure 1(a). Diffraction pattern produced by a grating. (b) Intensity profile of the diffraction pattern. It is plotted with the help of the software ImageJ. Figure 2. Pattern formed by the grating when there is no separation between the source and the grating. It has been reported (1) that bioluminescence emission from specimens of the Indian species of the firefly has the peak at 562 nm with full width at half maximum (FWHM) 55 nm. It has been inferred in a recent paper (2) that the firefly emission has a tendency for spectral narrowing within the narrow yellow sector of the spectrum. The present work conclusively establishes that statement. We propose that the firefly emits coherent light around the peak wavelength of 562 nm. References Gohain A, Barua S, Hazarika NM, Saikia GD. Baruah: Bioluminescence emissions of the firefly Luciola praeusta Kiesenwetter 1874 (Coleoptera: Lampyridae: Luciolinae), Journal of Biosciences 2009;34:287–92. Dehingia N, Baruah D, Siam C, Gohain A, Barua GD. Baruah: Purkinje effect and bioluminescence of fireflies, Current Science 2010;99:1425–7. Scrutinizing the oxidative and antioxidant properties of biologically active species: Fundamentals of the chemiluminescence approach Daniela I. Batovskaa, Galina F. Fedorovab, Vessela D. Kanchevaa, Valery A. Menshovb, Vladimir V. Naumovb, Alexey V. Trofimovb, Yuri B. Tsaplevb, Rostislav F. Vasil'evb and Timur L. Veprintsevb aInstitute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, ul. Acad. Bonchev, bl. 9, Sofia 1113, Bulgaria bEmanuel Institute of Biochemical Physics, Russian Academy of Sciences, ul. Kosygina 4, 119334 Moscow, Russian Federation E‐mail: avt_2003@mail.ru Keywords: chemiluminescence; free radicals; oxidation; bioantioxidants The emission of light followed the excited‐state generation in vitro (chemiluminescence) or in vivo (bioluminescence) is of prime interest for both pure and applied chemistry and biochemistry. In this context, oxidation processes constitute notable chemical generators of electronically excited light emitters. To convey a decisive role of oxygen, the latter phenomenon is sometimes called oxy‐chemiluminescence (1). Besides, biologically active free radicals which enter the human organism with environmental pollutants and tobacco smoke are prone to generating the electronically excited products. Our new advances in understanding the mentioned free‐radical processes enable developing the novel oxy‐chemiluminescence methodology to scrutinize the reactivity and damaging potential of the biologically active oxidants. Conversely, elucidation of the salient mechanistic features of the oxy‐chemiluminescence generation in the presence of the natural phenolic compounds is of importance for the assessment of their bioantioxidant propensity, most prominently of their ability to protect the cell‐membrane lipids against oxidative degradation. Our assay is based on the competition between disproportionation of peroxy radicals of the model hydrocarbon giving rise to light emission and scavenging the peroxy radicals by bioantioxidants resulting in the light quenching (1,2). The extent of quenching and the kinetics of the emission recovery upon bioantioxidant consumption depend on its reactivity towards peroxy radicals and concentration. Addition of natural bioantioxidants studied in this work to the probe oxy‐chemiluminescent “cocktails” not merely affects the light intensity through radical scavenging, but also results in the new excited‐state generation channels (3). The examples under present scrutiny range from technogenic air‐borne antioxidants to natural bioantioxidants of both vegetable and animal origin. Acknowledgments Generous support through the Bulgarian‐Russian bilateral academic program is gratefully appreciated. References Fedorova GF, Trofimov AV, Vasil'ev RF, Veprintsev TL. Peroxy‐radical‐mediated chemiluminescence: mechanistic diversity and fundamentals for antioxidant assay. ARKIVOC 2007;8:163–215. Fedorova GF, Menshov VA, Trofimov AV, Vasil'ev RF. Facile chemiluminescence assay for antioxidative properties of vegetable lipids: fundamentals and illustrative examples. Analyst 2009;134(10):2128–34. Vasil'ev RF, Kancheva VD, Fedorova GF, Batovska DI, Trofimov AV. Antioxidant activity of chalcones: the chemiluminescence determination of the reactivity and the quantum chemical calculation of the energies and structures of reagents and intermediates. Kinetics and Catalysis 2010;51(4):507–15. Fluorescence spectra of discharged photoprotein obelin at different calcium concentrations NV Belogurovaa and NS Kudryashevaa,b aInstitute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia. Fax: +7 391 2433400; tel: +7 391 2494242; E‐mail: nbelogurova@mail.ru bSiberian Federal University, Krasnoyarsk, 660043, Russia. Fax: +7 391 2448781; tel: +7 391 2445469; E‐mail: n_qdr@yahoo.com Photoprotein obelin, the enzyme‐substrate complex of polypeptide with 2‐hydroperoxycoelenterazine, is responsible for bioluminescence of hydroid Obelia longissima (1). Addition of Ca2+ to obelin triggers an oxidative decarboxylation of coelenterazine resulting in light emission with λmax = 485 nm. The product of bioluminescent reaction – enzyme‐bound coelenteramide called ‘discharged’ obelin – is a fluorescent protein. As the discharged obelin is a stable and nontoxic complex of polypeptide with chromophore molecule, it can be used in living cells as a fluorescent marker. Fluorescence spectra of discharged obelin (and hence, light color) are variable; they might depend on external physicochemical conditions – pH, temperature, calcium concentration. Change of the spectra might result from conformational transitions in the discharged obelin. The purpose here was to study spectra of obelin discharged at different (Ca2+). Fig. 1 shows the fluorescence spectra of obelin discharged at low and high (Ca2+) (1 and 3, respectively). The differences in these spectra are evident. Variation in (Ca2+) from 10−7 to 10−3 M revealed considerable spectral changes at (Ca2+) ≈ 6 • 10−7 M, this pointing to enzymatic conformational transition in photoprotein obelin (2). Removing of Ca2+ from obelin discharged at high (Ca2+) (spectrum 3) led to the formation of demineralized discharged obelin (2, Fig.1). Its spectrum is closer to that of obelin discharged at high (Ca2+) as compared to that discharged at low (Ca2+). Addition of Ca2+ to the demineralized obelin had no effect on its spectrum at different (Ca2+). Thus, demineralized obelin is a stable conformational structure independent of (Ca2+). It is highly probable that the dependence of discharged obelin fluorescence on (Ca2+) is conditioned by consecutive filling up of photoprotein Ca2+‐binding sites. Meanwhile, the results demonstrate that the enzymatic conformational transition in obelin is not reversible; removing of Ca2+ does not lead to significant change of fluorescence spectrum. We observed no changes in obelin bioluminescence spectra under different (Ca2+). The fact suggests that only one conformational structure of obelin is responsible for bioluminescence at various (Ca2+). The spectral changes were explained with regard to protonic interactions in the enzyme active center and attributed to change in acidity of the photoprotein chromophore (coelenteramide) in its fluorescent state S*1 (3). Figure 1. Fluorescence spectra, λexct = 330 nm: 1 − Ca2+‐discharged obelin, (Ca2+) = 3 · 10−7 M; 2 − demineralized discharged obelin, (Ca2+) = 0; 3 − Ca2+ − discharged obelin, (Ca2+) = 3 · 10−5 M; (obelin) = 3 · 10−7 M. References Vysotski ES, Lee J. Ca2+‐regulated photoproteins: structural insight into the bioluminescence mechanism, Acc. Chem. Res. 2004;37:405–15. 2. Belogurova NV, Kudryasheva NS. Discharged photoprotein obelin: fluorescence peculiarities // J.Photochem. Photobiol.B. 2010;101:103–8. Belogurova NV, Kudryasheva NS, Alieva RR, Sizykh AG. Spectral components of bioluminescence of aequorin and obelin. J. Photochem. Photobiol. B. 2008;92:117–22. Study of mechanisms of singlet oxygen generation by energy transfer processes from excited quantum dots Iryna V. Berezovska, Mykola M. Rozhitskii Kharkiv National University of Radio Electronics, 14, Lenin Ave, 61166, Kharkiv, Ukraine E‐mail: berezovskaya.irina@gmail.com, rzh@kture.kharkov.ua The achievements of nanotechnology are used in the treatment of cancer, specifically in photodynamic therapy (PDT). The method uses the light source and photosensitizer (Quantum Dots), which can be accumulated in the tumor. The PDT method is based on the physical processes occurring during the interaction of a photosensitizer and light with subsequent generation of singlet oxygen 1O2. This is type II photodynamic process. The process includes energy transfer from a triplet photosensitizer to triplet oxygen in a spin‐allowed process. The processes involved in this method are rather complex thus the study of their mechanisms is necessary. It is important to determine efficiency of quantum dots application for singlet oxygen generation. One of the methods for detecting singlet oxygen is the chemical traps method. The method is based on the optical bleaching of N,N‐dimethyl‐4‐nitrosoaniline (RNO) at 440 nm (1) caused by the product of O2 reaction with histidine (2) used as selective acceptor of 1O2, as shown in the following reaction schemes: S0+hν→k0S1, S1→k1T, T+O23→k3O21+S0, O21+G→k5GO2, GO2+RNO→k6−RNO+P, where S0, S1, T – ground, excited and triplet state of the photosensitizer (QD) respectively; 1O2, 3O2 – singlet and triplet (ground) state of the oxygen respectively; G – histidine; GO2 – transannular peroxide; RNO ‐ N,N‐dimethyl‐4‐nitrosoaniline; P – products. The bleaching of RNO is observed at 440 nm using a spectrophotometer as a result of singlet oxygen generation. Thus we can conclude about energy transfer from excited quantum dots to triplet oxygen. The abovementioned reactions can be modeled using the following kinetic equations: dS0dt=−k0S0+k3TO23, dS1dt=k0S0−k1S1, dTdt=k1S1−k3TO23, dO21dt=k3TO23−k5O21G, dGO2dt=k5O21G−k6GO2RNO The results of numerical simulation allow studying the consumption of oxygen in various states, and predicting the behavior of singlet oxygen reaction with transannular peroxide. Thus the results of theoretical and experimental investigation of reactions involved in PDT method show the applicability of quantum dots as efficient photosensitizers that can replace common organic dyes used for PDT. References Yan L. Magnetic Field Effects on Photosynthetic Reactions, printed by Print Partners Ipskamp B.V., the Netherlands 2008;113. Gomes AJ, Lunardi1 CN, Gonzalez S, and Tedesco AC. The Antioxidant Action of Polypodium Leucotomos Extract and Kojic Acid: Reactions with Reactive Oxygen Species, Braz J Med Biol Res 2001;34:1487–94. New Nanophotonic Detection Method of Carcinogenic Polycyclic Aromatic Hydrocarbons by the Example of Benzo(a)pyrene Olga A. Sushko, Olena M. Bilash and Mykola M. Rozhitskii Laboratory of Analytical Optochemotronics, Kharkiv National University of Radio Electronics,14 Lenin Ave, 61166, Kharkiv, Ukraine E‐mail: rzh@kture.kharkov.ua Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals formed during the incomplete burning of coal, oil, gas, wood, garbage or other organic substances such as tobacco and charbroiled meat. There are more than 100 different PAHs which are used in medicine and for production of dyes, plastics, pesticides ect. Also PAHs are contained in asphalt used in road construction, in crude oil, coal, coal tar pitch, creosote and roofing tar. PAHs are found throughout in air, water and soil. In air PAHs can form complexes with dust particles while in water, soil, solid sediments PAHs can exist as separate non‐soluble molecules. PAHs can be transformed by photochemical and/or chemical reaction to long‐living product with life‐time from days to weeks (1). Benzo(a)pyrene (BP), C20H12, is a five‐ring PAH which metabolites are mutagenic and highly carcinogenic. This means that BP is a procarcinogen the mechanism of BP carcinogenesis depends on its enzymatic metabolism to the ultimate mutagen, benzo(a)pyrene diol epoxide. The last intercalates into DNA, bonding covalently to the nucleophilic guanine bases (2). Therefore the very important problem is the definition of organic polyaromatic carcinogens, the most hazardous among which is BP, in water and other objects by efficient and cheap methods and instruments. There are a number of methods for the PAHs determination in water including high‐performance liquid chromatography, immuno‐chemical analysis, chemical and biological test methods (3). But these methods have several disadvantages, including complexity and high cost of the equipment, sample preparation and analysis procedure, not enough detection limit and selectivity, rather high cost and long assay duration. So the development of novel methods and instruments for the definition of low content of carcinogenic polyaromatic compounds is quite urgent task. Above‐mentioned disadvantages are practically absent in the proposed nanophotonic assay method and sensor device. The nanophotonic method under consideration is based on electrochemiluminescent analysis and modern nanomaterials – quantum‐dimensional semiconductor structures used in the developed nanophotonic sensor's device. The developed sensor itself represents a very small by its dimensions thin layer cell with two or more electrodes intended both for electrochemical and luminescent assays. The working electrode surface inside the sensor's active volume is being modified by Langmuir‐Blodgett or spin‐coating methods with quantum‐dimensions structures such as quantum dots or quantum tubes used as detector elements. The investigation of the developed method and sensor's device show high performance and metrological characteristics such as a low detection limit (< 1 nM), low assay duration and cost, high selectivity and reproducibility. The authors gratefully acknowledge the support for this research by Science and Technology Center in Ukraine Project 5067 (Project Manager: Prof. Rozhitskii M.M). References Moiz Mumtaz, Julia George. Toxicological Profile for Polycyclic Aromatic Hydrocarbons. U.S. Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry. 1995;246–9. Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hotspots in P53. Science. 1996 October 18;274(5286):430–2. Bilash OM, Galaichenko OM, Sushko OA, Rozhitskii MM. “New nanophotonic detection method of benzo(a)pyrene. Scientific Council on ″Analytical Chemistry”. Ukraine. 2011 May 16 – 20. 58. A study of neonothopanus nambi luminescent system VS Bondar, AP Puzyr, KV Purtov, SE Medvedeva, EK Rodicheva, GS Kalacheva and JI Gitelson Institute of Biophysics, SB RAS, Akademgorodok, Krasnoyarsk, 660036 Russia Siberian Federal University, Krasnoyarsk, 660041 Russia E‐mail: bondvs@mail.ru There are several higher mushrooms that emit visible light (1,2), however, up to the present little is known about their luminescence mechanism, and there is no unambiguous opinion about molecular organization of their luminescent systems (3,4). The luminescent system from Neonothopanus nambi luminous fungus found by Dao Van in tropical forests of Southern Viet Nam and initially described as Omphalotus af. illudent species (5) was investigated. Mycelium was grown in liquid nutrient potato‐sucrose medium at 26 °C for 8–10 days. Bioluminescence of mycelium was measured with BLM 8801 luminometer («Nauka» Special Design Bureau, Krasnoyarsk, Russia), calibrated against Hasting‐Weber standard (6) (one unit is 108 photons in 1 second). Components of nutrient media prior and after growth of mycelium and fungal extracts were analyzed with Agilent 5975Inert mass‐spectrometer (Agilent, USA). Mycelium of N. nambi has been shown to exhibit long‐lasting bioluminescence. Time to maximum is from 40 minutes to 5 hours. After that the luminescence slowly decreases and reaches steady‐state level maintained for 10–12 hours. The luminescence spectrum of N. nambi was shown to be in the visible range (wavelength range 480–700 nm) with maximum 527–535 nm. Luminescence of N. nambi mycelium was shown to be stimulated by additions of hydrogen peroxide. This may indicate participation of peroxidases in the bioluminescence mechanism of this mushroom. As Mn ions stimulate luminescence of N. nambi mycelium, the composition of lignin‐braking enzymic complex of the mushroom can be expected to comprise Mn‐peroxidase (or Mn‐peroxidases). Different methods of breaking N. nambi mycelium and drying of mycelium and fruiting bodies have been shown to result in irreversible loss of luminescence. These data count in favor of localization of the mushroom luminescent system on the cell membrane or other cell structures. The mushroom extracts have been found to have thermostable compound which increases its bioluminescence by several orders and might be the emitter of light. Comparative analysis of chemical composition of nutrient media (before and after cultivation of N. nambi mycelium) showed a large amount of phenolic compounds released into the medium during growth of the mushroom. The composition of fatty acids of the mycelium grown in different media was found to have hydroxy acids with different position of the ‐OH groups. Luminescence of N. nambi mycelium grown on rich medium increases by several orders after its incubation in deionized water. High radiance can persist for a long time. Comparative analysis of fatty acid composition of the mycelium in the rich medium exposed to deionized water indicate activation of peroxidation of lipid components of the mushroom in the lean medium. Experimental data allow assuming that the probable mechanism of higher mushrooms' luminescence is the chemiluminescent reaction which may be provided for by functioning of two enzymic systems – peroxidases of lignin‐breaking complex and membrane‐bound system of cytochrome P450 catalyzing oxidation of the organic substrate with participation of active forms of oxygen. This work was supported: by the Federal Agency for Science and Innovation within the Federal Special Purpose Program (contract No 02.740.11.0766), by the Program of the Government of Russian Federation «Measures to Attract Leading Scientists to Russian Educational Institutions» (grant No 11. G34.31.058). References Herring PJ. Mycologist. 1994;8:181–3. Vydryakova GA, Psurtseva NV, Belova NV, Pashenova NV, Gitelson JI. Mycology and Phytopathology. 2009;43:369–75. Shimomura O. Bioluminescence: chemical principles and methods. World Sci. Publ.Co. Pte. Ltd., 2006;266–300. Desjardin DE. Oliveira AG, Stevani CV. Photochem. Photobiol. Sci. 2008;7:170–82. Dao TV. Biotechnology. 2009. №6. P.74–8. Hastings JW, Weber G. J. Opt. Soc. Am. 1963;53:1410–5. The half‐reactions of firefly luciferase bioluminescence proceed via the domain alternation mechanism Bruce R. Branchini*, Justin C. Rosenberg, Danielle M. Fontaine, Tara L, Southworth, Curran E. Behney and Lerna Uzasci Department of Chemistry, Connecticut College, New London, CT 06320, USA *E‐mail: brbra@conncoll.edu According to the domain alternation mechanism, the large “ANL” superfamily of enzymes that includes the acyl‐CoA synthetases and nonribosomal peptide synthetases (NRPSs) catalyze adenylate‐ and thioester‐forming half‐reactions in two different conformations related by an ~140 degree domain rotation of the C‐domain (1). The beetle luciferases are members of the ANL superfamily and likewise initiate the bioluminescence process by converting firefly luciferin into the corresponding adenylate. However, the second luciferase catalyzed half‐reaction is a mechanistically dissimilar oxidative process that produces light. We have demonstrated by LC/ESI‐MS, SDS‐PAGE and Ellman's (sulfhydryl) analysis that a luciferase variant constructed for this study containing Cys residues only at surface positions 108 (N‐Domain) and 447 (C‐Domain) can be intramolecularly cross‐linked with the bifunctional reagent 1,2‐bis(maleimido)ethane (BMOE) (2). Proteolytic sequencing and LC/ESI‐MS analysis verified the existence of a new peptide containing the BMOE moiety that could only arise from the intramolecularly cross‐linked luciferase protein in a domain‐rotated conformation predicted by the alternation mechanism, but previously undocumented in structural studies (3, 4). The trapped luciferase conformer retains catalytic activity for only the 2nd half‐reaction and this conformer is very likely the one in which photon production occurs. While we have characterized a conformer that is apparently similar to that adopted by the thioester‐forming superfamily enzymes, additional x‐ray crystallographic studies are in progress to further structurally characterize the chemically trapped luciferase. Additional studies related to issues of bioluminescence color determination will also be discussed. References Gulick AM. Conformational Dynamics in the Acyl‐CoA Synthetases, Adenylation Domains of Non‐ribosomal Peptide Synthetases, and Firefly Luciferase, Acs Chemical Biology 2009;4:811–27. Branchini BR, Rosenberg JC, Fontaine DM, Southworth TL, Behney CE, Uzasci L. Bioluminescence Is Produced from a Trapped Firefly Luciferase Conformation Predicted by the Domain Alternation Mechanism, JACS 2011;133:11088–91. Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate‐forming enzymes, Structure 1996;4:287–98. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence, Nature 2006;440:372–6. Sensitivity of Ca2+‐regulated photoprotein bioluminescence to magnesium ions is determined by EF‐hand motif III LP Burakova, NP Malikova and ES Vysotski Photobiology Lab, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia E‐mail: burakoval@mail.ru The Ca2+‐regulated photoprotein is a complex of a single chain polypeptide and a peroxy‐substituted coelenterazine (2‐hydroperoxycoelenterazine), which is tightly, though noncovalently, bound to the polypeptide. Bioluminescence initiated by Ca2+ results from oxidative decarboxylation of 2‐hydroperoxycoelenterazine. All Ca2+‐regulated photoproteins belong to EF‐hand protein family and have the similar spatial structures. The mainstream applications of Ca2+‐regulated photoproteins take advantage of their inherent property to emit light on calcium binding. Owing to this property and because photoproteins are highly sensitive for detecting calcium, they have been widely used as probes of cellular Ca2+. From the standpoint of intracellular measurements, free magnesium ion concentration is the most important factor known to influence the sensitivity of photoproteins to Ca2+. In the case of aequorin, Mg2+ within the range of concentrations (in the vicinity of 1 mM) that likely might be encountered inside living cells, reduces Ca2+‐independent luminescence (1) and sensitivity to calcium (2), i.e. shifts the Ca2+ concentration‐effect curve to the right. However, it may be not so critical for other photoproteins because for obelin, for example, the effect of Mg2+ is much less pronounced than for aequorin bioluminescence (3). In this study using chimeric Ca2+‐regulated photoproteins consisting of different EF‐hand motifs of obelin and aequorin we show that EF‐hand motif III is responsible for sensitivity or insensitivity of photoprotein bioluminescence to magnesium ions (Figure 1). Figure 1. Ca2+ concentration‐effect curves with 1 mM Mg2+ (green triangles) and without Mg2+ (red circles) for OL(I)‐AV(II‐III)‐OL(IV) (left ) and AV(I)‐OL(II‐III)‐AV(IV) (right). Filled symbols, (Ca2+) was obtained with Ca2+–EGTA buffers. Unfilled symbols, (Ca2+) was obtained with dilution of CaCl2 solution. L, luminescence intensity at fixed (Ca2+); Lint, integral light in saturating (Ca2+). References Ray BC, Ho S, Kemple MD, Prendergast FG, Nageswara Rao BDN. Biochemistry 1985;24:4280–87. Hastings JW, Mitchell G, Mattingly PH, Blinks JR, van Leeuwen M. Nature 1969;222:1047–50. Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee J. Biochemistry 2002;41:2227–36. pH sensitivity of color shift of firefly chromophore due to electrostatic field from neighboring water‐ions Duanjun Caia,b*, Miguel AL. Marquesb,c and Fernando Nogueirab aDepartment of Physics, Xiamen University, Xiamen 361005, China bCFC, Departamento de Física, Universidade de Coimbra, 3004‐516, Coimbra, Portugal cLaboratoire de Physique de la Matière Condensée et Nanostructures, Université Lyon I, CNRS, UMR 5586, Domaine scientifique de la Doua, F‐69622 Villeurbanne Cedex, France *E‐mail: dcai@xmu.edu.cn Firefly luciferase, famous example of fluorescent proteins, has been successfully applied in various bio‐imaging techniques, as a reporter for ATP generation, gene expression, and biosensors for environmental pollutants (1,2). Issues on fluorescent colors, e.g., color shift, color tuning, and color brightness (3), are intensively concerned aiming at extensive color modulation for special marking demands. Among these studies, one of the most frequently used chemical factors, pH value, was found having distinct influences on the color shift and spectral deformations. It has been widely reported that red shift of luminescence occurs with lowering pH, whereas blue shift comes up at higher pH (4). In particular, quantitative re‐examination of the quantum yield of firefly bioluminescence showed that the spectral lineshape variation could be the real reason for the color change. However, in‐depth mechanism of these interesting phenomena linked with pH remains ambiguous and explanations exclusive. In general, the pH value represents the macroscopic activity of water ions, i.e., hydrogen ions and hydroxide ions. While, in fact, the direct pH effect usually works through the relatively local interaction between these active ions and the host molecule in solvent. Recently, it has been observed that the color shift of firefly is associated with the surrounding electrostatic fields (5). This suggests that in the protein pocket embracing trace water, the location and electrostatic field of H+ and/or OH− can be critically associated with the color shift at different pH. Despite the “Zundel” or “Eigen” model,6 in trace water environment the preferable hydronium ion structure should still be H3O+. The electrostatic fields generated by neutral water ions were calculated by Octopus code, as depicted in Figure 1b‐d. It is found that due to the polarized orientation and hydrogen‐bond coordination, the surrounding electric fields of water, H3O+ and OH− show different shapes of butterfly‐like, dumbbell‐like and dual‐peach‐like, respectively. The negative electric field from H2O merely exists within a radius of a few angstroms and then drops rapidly, indicating a short rang of electrostatic interaction. This shows the orientation dependence of electrostatic interaction between water and nearby objects, e.g., molecules. In contrast, the H3O+ ion generates a positive field and the electrical working distance, as long as 6~10 Å along x and y, is much longer than that of the neutral H2O. Figure 1. (a) Firefly protein structure with chromophore OxyLH2 and water molecules in the pocket. (b), (c) and (d), electrostatic distribution of H2O, H3O+ and OH−, respectively, showing shape of butterfly‐like, dumbbell‐like and dual‐peach‐like. The sector angles indicating the area of field absence and the interaction region are marked. Water molecules, hydronium and hydroxyl ions were introduced to the preferable active sites close to OxyLH2 inside the protein cavity, as shown in Figure 1a, where the most important sites have been found to be # 2325 and 2470 at two ends of the molecular long axis that is along the polarization orientation. The geometries (including hydrogens) of H2O, H3O+, OH− and OxyLH2 were optimized and the final excitation spectra of OxyLH2 are plotted in Figure 2. Because of the weak electric field generated by the neutral water molecules (Figure 1b), it can be seen that the influences on the color or spectral shape are negligibly small (slightly blue shift, < 0.05 eV). The H3O+ existing in a lower pH environment prefers the residence at # 2325 site and the positive electrostatic interaction leads to an obvious red shift by about 0.2 eV On the contrary, the OH‐ 2470 ion at higher pH locates on the side close to the deprotonated O in the benzothiazole ring (Figure 1a), which results in a significant blue shift of A1 by 0.27 eV with respect to the emission in neutral solvent, accompanying with strong enhancement of emission intensity by 24%. The strong pH sensitivity of subpeak intensity below pH 8.0 as observed in Ref. 3 could be explained, except that here we find that the blue shift actually stems from the energetic shift as well as increase of peak intensity, rather than simple intensity variation. Figure 2. Excitation spectra of OxyLH2 in the protein pocket containing critical surrounding water molecules (H2O 2325 and 2470) and/or their ion states. We thank the CFC of the University of Coimbra and the Milipeia supercomputer system for providing CPU time. References Viviani VR. Cell. Mol. Life Sci. 2002;59:1833–50. Chalfie M, Tu M, Euskirchen G, Ward WW, Prasherf DC. Science 1994;263:802–5. Branchini BR, Southworth TL, Murtiashaw MH, Magyar RA, Gonzalez SA, Ruggiero MC, Stroh JG. Biochemistry 2004;43:7255–62. Branchini BR, Ablamsky DM, Rosenman JM, Uzasci L, Southworth TL, Zimmer M. Biochemistry 2007;46:13847–55. Cai DJ, Marques MAL, Nogueira F. J. Phys. Chem. B 2011;115:329–32. Zundel G, Metzger HZ. Physik. Chem. (N.F.) 1968;58:225–45. Electrochemiluminescence determination of an antihistamine pharmaceutics, doxylamine succinate Roshanak Ghobadiana, Mohammad Javad Chaichia*, Mohammad Reza Ganjalib, Parviz Norouzib and Morteza Hosseinic aFaculty of Chemistry, University of Mazandaran, Babolsar, Iran bCenter of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran cDepartment of life science engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran *E‐mail: jchaichi@yahoo.com (M. J. Chaichi) Electrochemiluminescence involves the generation of species at electrode surfaces that then undergo electron‐transfer reactions to form excited states that emit light (1). Doxylamine is a member of the ethanolamine class of antihistamines and has anti‐allergy power, branded as Unisom. Various analytical procedures have been adopted to determine doxylamine succinate (such as (2, 3)). Ru(bpy )32+ ECL has been widely used as a detection method for various pharmaceuticals and antibiotics, when the analytes often do not contain a good chromophore (4). Also with this method pharmaceuticals can be determine without prior derivatisation, having wide dynamic range and low detection limit. Doxylamine succinate contains a tertiary amine functional group that can be determined by utilizing its electrogenerated chemiluminescence reaction with (Ru(bpy)32+) by proposed mechanism shown in equations (1–5). Rubpy32+→Rubpy33++e– (1) RCH 2 N(CH 3)2 → RCH 2 N + •(CH 3)2 + e – (2) RCH 2 N + •(CH 3)2 → RCHN •(CH 3)2 + H + (3) RCHN•CH32+Rubpy33+→+Rubpy32+*+oxidizedproduct (4) Rubpy32+*→Rubpy32++hv (5) In this work the ECL reaction described above has been used to determine doxylamine succinate by a homemade instrument and the amounts of doxylamine succinate were quantified in the human urine as an application of proposed method. A calibration was carried out for doxylamine succinate utilizing the optimized conditions. A concentration range of 1 × 10‐6 −1 × 10−4 mol l−1 for doxylamine succinate was analyzed and each standard contained 5mM Ru(bpy)32+. The effect of pH on the ECL response of Ru(bpy)3 2+/doxylamine succinate system was studied by using various pH phosphate and acetate buffer solutions. Maximum ECL intensity was obtained in pH 7.5 PBS. ECL response significantly correlates with the oxidation of the Ru(bpy)32+, that depends on applied potential to the working electrode. The maximum ECL response was observed at 1.3 V. For the 1 mM Ru(bpy)32+, 0.15 mM Doxylamine succinate in a pH = 7.5, 0.15 mol L−1 phosphate buffer solution, ECL signal was zero for potential lower than 1.0 V (vs. Ag/AgCl). At higher potentials, signal was increased up to potential 1.3 v (vs. Ag/AgCl), and at higher potential there was a decreasing in the ECL response, possibly due to the negative effect of the oxidation of water. The effect of Ru(bpy)32+ concentration on ECL intensity is studied (Fig. 1). In the Ru(bpy)32+/ doxylamine succinate system, ΔI was increased greatly with increase of Ru(bpy)32+ concentration in the range 1 × 10−6 to 1 × 10−3 mol L‐1, and above 1 × 10 −3 ΔI decreasing was observed, after 3×10−3 ΔI became constant. So 1 × 10 −3 mol L−1 of Ru(bpy)32+ was chosen in order to obtain a high signal to noise ratio. ECL response was increased, with increase of doxylamine succinate concentration (Fig. 2) in the optimized experimental conditions. A straight line obtained in the investigated concentration range as shown in Fig. 2, is represented by the equation y = 5 × 106x + 107.8. Where y is the ECL intensity (mV) and x the doxylamine succinate concentration (molL−1). The linear ECL response to concentrations of doxylamine succinate was from 1 ×1 0−6 to 3 × 10 −4 mol L−1 with a correlation coefficient of 0.999 and a detection limit of 0.3 μgL−1. The precision of the method was studied assaying three concentration levels: 0.02, 0.1, 0.3 mmolL−1 doxylamine succinate. The RSD (n = 5) amounts were 2.6, 1.88, 0.97 % respectively. Each urine sample was spiked with doxylamine succinate over the rang from 6 × 10 5 to 4.5 × 10−4 mol L−1. The results showed that there was no significant interference for signal achievement by proposed method. The recovery of method was %98.16. Figure 1. Ru(bpy)32+ concentration effect on the ECL intensity in solution containing 0.15mM Doxylamine succinate in a pH = 7.5,0.15 molL−1 phosphate buffer solution, scan rate 100mvs−1. Figure 2. Calibration curves for standards concentration of doxylamine succinate in 0.15 mol l−1, pH = 7.5 phosphate buffer solution, scan rate100mvs‐1. The inset shows ECL peaks at different concentration of the drug with two replicates. In conclusion the proposed method could be used for quantification of pharmaceuticals without needs to derivative and is applicable for pharmaceuticals without chromophore group. Also, this method could be used to monitor doxylamine succinate and other pharmaceuticals containing tertiary amine groups in urine. References Mark M. Richter. Chem. Rev. 2004;104:3003–36. Pathak A, Rajput SJ, Journal of AOAC International Y. 2008;91:1059–69. Li Monferrer‐Pons, J.S. Esteve‐Romero, G. Ramis‐Ramos, M. C. Garcia‐Alvarez‐Coque, Analytical Letters 1996;29:1399–13. Inês N. Tomita, Luis O.S. Bulhões, Analytica. Chimica. Acta 2001;442:201–6. Chemiluminescence characteristics of quinoxaline derivative as green fluorophores in peroxyoxalat‐hydrogen peroxide system MJ Chaichi*, A Khodabandeh, R Akhoondi, T Khajvand and MR Sadeghi Maleki Faculty of Chemistry, University of Mazandaran, Babolsar, Iran Peroxyoxalate chemiluminescence (PO‐CL) is well known as a powerful means of detecting various fluorophores and hydrogen peroxide. PO‐CL is based on the reaction of H2O2 with peroxyoxalate which result an intermediate (1). The excited intermediate transfers its energy to an efficient fluorophore through the chemically initiated electron exchange luminescence (CIEEL) mechanism (2). Quinoxaline derivatives are of great interest as fluorescent emitters for peroxyoxalate chemiluminescence. These compounds have been utilized as fluorescence probes in some of elaborated chemosensors (3,4). In this investigation, the new quinoxaline derivative (Figure 1) used as a fluorophore, which produce a green light in the chemiluminescence system. The relationship between the chemiluminescence intensity and concentrations of fluorophore, peroxyoxalate, sodium salicylate and hydrogen peroxide was investigated. Kinetic parameters for the peroxyoxalate‐chemiluminescence were also calculated from the computer fitting of the corresponding chemiluminescence intensity/time profile. It was found that the new quinoxaline derivative can be used as an efficient green fluorescent emitter. Figure 1. Chemical structure of the quinoxaline fluorophore. References Chaichi MJ, Karami AR, Shockravi A, Shamsipur M. Chemiluminescence characteristics of cumarin derivatives as blue fluorescers in peroxyoxalate‐hydrogen peroxide system. Spectrochim. Acta Part A 2003;59:1145–50. Schuster GB. Chemiluminescence of organic peroxides. Conversion of ground‐state reactants to excited‐state products by the chemically initiated electron‐exchange luminescence mechanism. Acc. Chem. Res. 1979;12(10):366–73. Yamamoto T, Sugiyama K, Kushida T, Inoue T, Kanbara T. Preparation of new electron‐accepting π‐conjugated polyquinoxalines. chemical and electrochemical reduction, electrically conducting properties, and use in light‐emitting diodes. J. Am. Chem. Soc. 1996;118(16):3930–7. Samadi‐Maybodi A, Akhoondi R, Chaichi MJ. Studies of New Peroxyoxalate‐H2O2 Chemiluminescence System Using Quinoxaline Derivatives as Green Fluorophores. J. Fluoresc. 2010;20:671–9. Chemiluminescence characteristics of furan derivatives as blue fluorescers in peroxyoxalate‐hydrogen peroxide system MJ Chaichia*, SN Azizia, M. Heidarpoura, O Aalijanpoura and M Qandaleeb aFaculty of Chemistry, Mazandaran University, Babolsar, I.R. Iran bDepartment of Biology, Garmsar Branch, Islamic Azad University, Garmsar, Iran *E‐mail: jchaichi@yahoo.com Furan derivatives are of great interest as fluorescent emitters for peroxyoxalate chemiluminescence (1). Reaction of peroxyoxalates such as bis‐(2,4,6‐trichloro‐phenyl) oxalate with H2O2 can transfer energy to fluorophore via formation of dioxetanedione intermediate (2). Furan derivatives used as a novel fluorescer in this study which produces a blue light in the chemiluminescence systems(Figure 1). The relationship between the chemiluminescence intensity and concentrations of TCPO, sodium salicylate, hydrogen peroxide and the fluorescer has been investigated. The linear ranges for furan derivatives A and B are 0.25–5 × 10−4 M and 0.1–5 × 10−4 M respectively. Kinetic parameters for the peroxyoxalate‐chemiluminescence are also calculated from the computer fitting of the corresponding chemiluminescence intensity/time profiles. Figure 1. Molecular structure of (A) Dimethyl 2‐((2,6‐dimethylphenyl)amino)‐5‐((E)‐ 2‐phenyl‐1‐ethenyl)‐3,4‐furan dicarboxylate (B) Dimethyl 2‐(t‐buthyl amino)‐5‐((E) 2‐phenyl‐1‐ethenyl)‐3,4‐furan dicarboxylate. Keywords: Peroxyoxalate chemiluminescence; fluorescer; TCPO; hydrogen peroxide; furan derivative References Asghari S, Qandalee M. Facile One‐Pot Synthesis of Amino Furans Using Trans‐Cinnamaldehyde in the Presence of Nucleophilic Isocyanides. Acta Chim. Slov. 2007;54:638–41. Samadi‐Maybodi A, Akhoondi R, Chaich MJ. Studies of New Peroxyoxalate‐H2O2 Chemiluminescence System Using Quinoxaline Derivatives as Green Fluorophores. J. Fluoresc. 2010;20:671–9. Indirect chemiluminescence‐based determination of catecholamines in pharmaceutical formulations by diethyl‐2‐(tertbutylamino)‐5‐((E)‐2‐phenyl‐1‐ethenyl)‐3,4 furandicarboxylate as a novel blue fluorescer in peroxyoxalate system Mohammad Javad Chaichia*, Tahereh Khadjvanda, Sakineh Asgharia and Mohammad Qandaleeb aAnalytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar 4741695447, Iran bDepartment of biology, Garmsar branch, Islamic azad university, Garmsar, Iran *E‐mail: jchaichi@yahoo.com Catecholamines are compounds that consist of amines attached to a benzene ring bearing two hydroxyl groups (catechol). The main sites of catecholamine production are the brain, chromaffin cells of the adrenal medulla and the sympathetic neurons (1). Because of the significance of catecholamines in clinical diagnosis and medical treatment, rapid and sensitive determination of these compounds in biological samples and pharmaceutical formulations is therefore very important. Various analytical methods have been developed for the determination of CAs, some of these are based on HPLC with FL and capillary electrophoresis (2,3). Recently, highly sensitive methods using HPLC‐mass spectrometry (MS) have been developed for the determination of CAs (4). Although these analytical methods employing HPLC‐MS or HPLC‐MS/MS afford high sensitivity, the cost of the equipment still limits. For the first time, we propose a novel highly fluorescent fluorescer diethyl‐2‐(tertbutylamino)‐5‐((E)‐2‐phenyl‐1‐ethenyl)‐3,4 furandicarboxylate for PO‐CL detection of CAs (5). The method was developed based on quenching effect dopamine (DA) and enhancing effect epinephrine (E) and methyldopa (MD) on TCPO–H2O2–SS–fluorescer CL system (figure 1). This decrease and increase was evaluated in relation to the original chemiluminescence emission corresponding to a blank and it was proportional to the DA, E and MD concentration (ΔCL). Under optimal conditions, good linear ranges were obtained, 0.5–12.7 µg/ml, 0.06–1.83 µg/ml and 0.069–3.52 µg/ml with detection limits of 0.30, 0.03 and 0.04 µg/ml (S/N = 3) for DA, E and MD, respectively. The relative standard deviations of five replicate measurements of DA, E and MD were 1.9, 2.5 and 5.2%, respectively. Figure 1. Time course of the kinetic profile of TCPO‐H2O2– fluorescer–SS system after mixing of (H2O2)= 1.3×10‐3 M, (SS)= 8.0×10‐4 M, (fluorescer)=3.3×10‐3 M and (TCPO)= 2.5×10‐3 M, (DA)= 3.3×10‐5 M, (E) and (MD)= 5.0×10‐6 M. The effects of each of the CAs on the chemiluminescence profile and reaction kinetics were determined under pseudo‐first‐order condition with hydrogen peroxide in large excess over TCPO and a pooled intermediate model was used (6). The fluorescence spectra of the TCPO–H2O2–flourescer reaction in the absence and presence of CAs had the same maximum emission wavelength at 420 nm more. The presence of CAs had no obvious difference compared with the fluorescence spectrum of fluorescer and the fluorescence intensity of fluorescer, which indicated no obvious intraction, occurred between fluorescer and CAs. Based on the CL and fluorescence spectrum and kinetic data, the crucial step in quenching the luminescence was assumed to be the reaction of the analytes with the highly energetic intermediate to give CL products, in competition with the reaction of fluorescence. Compared with other common methods, the proposed method is simple, cheap, sensitive, rapid, and suitable for analysis and be applied to the determination of those three CA derivatives in their pharmaceutical preparations with satisfactory results. References Whitley RJ, Meikle AW, Watts NB. Endocrinology, in: Burtis CA, Ashwood ER (Eds.), Tietz Textbook of Clinical Chemistry, 2nd ed., Saunders WB, Philadelphia, 1994;1739. Zydroń M, Baranowski J, Białkowski J, Baranowska I, Separation Science and Technology 2005;40:3137–48. Liu YM, Wang CQ, Mu HB, Cao JT, Zheng YL. Electrophoresis 2007;28:1937–41. Gu Q, Shi X, Yin P, Gao P, Lu X, Xu G. Analytica Chimica Acta 2008;609:192–200. Asghari S, Qandalee M. Acta Chim. Slov. 2007;54:638. Dye JL and Nicely VA. J. Chem. Educ. 1971;48:443–8. Optimization of chemiluminescence based on hydrogen peroxide–sodium hydrogen carbonate–CdS quantum dots system using Box‐ Behnken Design SN Azizi, MJ Chaichi* and Parmis Shakeri Analytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran *E‐mail: jchaichi@yahoo.com Semiconductor nanocrystals known as quantum dots (QDs) are in high‐demand as inorganic fluorophores. Compared to traditional organic fluorophores, they offer several advantages, including flexible photoexcitation, sharp photoemission, and superb resistance to photobleaching (1). Additionally, by changing the size or composition of the nanocrystals, their optical properties can be tailored to meet specific wavelength requirements. Luminescent properties of semiconductor nanocrystals are usually investigated by photoluminescence (PL) produced using photoexcitation 'electrochemiluminescence (ECL) generated by electron injection and cathodoluminescence given from electron impact. However, chemiluminescence (CL) generated from chemical energy excitation is rarely used to study the luminescent property of semiconductor nanocrystals (2). The CL property of CdSe/CdS core/shell nanostructure dealing with the emitting state related to the quantum‐confined orbitals. The CL of CdTe NCs and CdS NCs directly oxidized by some oxidants, such as H2O2 and KMnO4, and its size‐dependent and surfactant‐sensitized effects (3). It was reported that peroxymonocarbonate ion (HCO4−) is a luminous species and can be generated in HCO3−–H2O2 system (4). But HCO4− provided a weak chemiluminescence emission, which can be enhanced in the presence of sensitizers or fluorophore compounds. Several compounds can be used, and special attention has been given to QDs due to their high quantum yields. Therefore in this study we investigate the effects of CdS QDs on the NaHCO3‐H2O2 CL system. The results show that the CL intensity of NaHCO3‐H2O2‐CdS NCs system is far stronger than that of NaHCO3‐H2O2 system, indicating the great sensitized effect of CdS NCs on NaHCO3‐H2O2 CL reaction. Parameters influencing the CL signals of NaHCO3‐H2O2‐CdS NCs system were then investigated systematically to establish the optimal conditions for the CL reaction. Because of many factors involved in the optimization of this analytical method, experimental design was utilized to make the developing process more efficient and cost‐effective. The optimum conditions for maximizing the CL emission intensity were determined by means of a Box–Behnken experimental design combined with response surface modeling (RSM) and quadratic programming. The possible CL mechanisms were proposed by means of the kinetic curves of CdS QDs in NaHCO3–H2O2 CL system, photoluminescence spectra, CL spectra, and transmission electron microscopy studies. This work is not only will be helpful to study physical chemistry properties of semiconductor nanocrystals but also are estimated to discover use in many fields such as luminescence devices, bioanalysis, and multicolor labeling probes. References Parak WJ, Pellegrino T, Plank C. Nanotechnology 2005;16:R9. Wang Zh, Li J, Liu B, Hu J, Yao X, Li J. J. Phys. Chem. B 2005;109:23304. Wang ZP, Li J, Liu B, Hu JQ, Yao X, Li JH. J. Phys. Chem. B 2005;109:23304. Yao HR, Richardson DE. J. Am. Chem. Soc. 2003;125:6211. Resonance rayleigh scattering technology for determination of nucleic acids at nanogram levels Yanjing Chen and Shuzhen Pan School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Nucleic acids are the essential material in the organism and have an important function in life process, Quantitative determination of nucleic acids was the base of investigation on nucleic acids (1,2). Resonance Rayleigh Scattering (RRS) technique has become a new interesting method for determination of micro amounts of biomacromoleculers with a lot of advantages such as high sensitivity, simplicity and quickness (3,4). In this paper, a new method for nucleic acids assay by RRS with scopoletine(SLT,7‐hydroxy‐6‐methoxylcoumarin) and surfactant CTMAB is developed. In the presence of CTMAB, the RRS signal of SLT can be greatly enhanced by nucleic acids, and the enhanced RRS intensity is in proportion to the concentration of nucleic acids, so nucleic acids at nanogram levels can be determined. The detection limits were 1.5 ng/mL for ctDNA, 3.6 ng/mL for hsDNA, 3.0 ng/mL for fsDNA and 2.5 ng/mL for yRNA. This method is proved to be convenient, rapid and highly sensitive. To a 10 mL colorimetric tube, solutions were added in the following order: 0.1 mL of 1.00 × 10−6 mol. L−1 SLT, 1.0 ml of C8H5KO4‐NaOH buffer, 0.8 mL of 1.00 × 10‐3mol. L−1 CTMAB and definite standard nucleic acids or sample solution. The mixture was diluted to 10 mL with water and allowed to stand for 15 min. All RRS spectra was obtained by scanning simultaneously with the same wavelength of excitation and emission in the range of 350–430 nm by spectrofluorometer with a 1.0 cm quartz cell.The intensity of RRS was measured at 393 nm with slit width at 10 nm for the excitation and 2.5 nm for emission. The enhanced RRS intensity of SLT‐CTMAB system by nucleic acids was represented as △IRRS=IRRS‐I0RRS, here IRRS and I0RRS were the intensities of the systems with and without nucleic acids. The RRS spectra of SLT, SLT‐CTMAB, SLT‐DNA and SLT‐CTMAB‐nucleic acids are shown in Fig. 1. It can be seen that the weak RRS of SLT can be enhanced by the addition of DNA. Moreover, when CTMAB is added into the system together with DNA, the RRS signal is greatly enhanced and the synergistic enhancement can be observed in the wavelength range 350–430 nm. The intensity of the RRS signal located at 393 nm is the strongest. Here, the 393 nm peak is chosen for further study. Figure 1. The RRS spectra. a: SLT; b: SLT+CTMAB; c: SLT+hsDNA; d: SLT+CTMAB+yRNA e: SLT+ CTMAB+hsDNA;. Conditions: SLT: 1.00 × 10−8 8mol/L; CTMAB:8.00 − 10−5 mol; Nucleic acids: 2 mg/mL; p H5.20. The optimum analytical conditions of the system were studied in a series of experiments. The experimental results indicate that △IRRS is enhanced obviously in the pH range 4.80–5.60, at pH 5.20 the △IRRS reached maximum, and 1.0 mL of C8H5KO4‐NaOH buffer is the most suitable for this assay. The effect of the concentration of SLT and CTMAB on the RRS signal of the system was investigated. The study shows that the maximum enhancement occurred when the concentration of SLT was 1.00 × 10−8 mol/L and that of CTMAB was 8.00 × 10−5 mol/L, respectively. The addition order of the reagents was selected as follows: SLT‐buffer‐CTMAB‐nucleic acid. Test showed that the RRS intensity reached a maximum within 10 min after the reagents had been added and remained stable for at least 3 h. A number of foreign substances including metal ions, amino acids and proteins were tested for their effects at hsDNA concentration 1.0 × 10−8 g/mL and the result showed that most substances did not interfere appreciably with the assay. Under the optimum conditions, the linear regression equations between △IRRS and the concentration of nucleic acids were established. All parameters are presented in Table 1. It can be seen that this method has a higher sensitivity than most of other RRS methods. Table 1. All analytical parameters Nucleic acid Linear range (g.mL‐1) r LOD (ng.mL‐1) hsDNA 1.0×10‐8‐7.0×10‐6 0.9990 3.6 ctDNA fsDNA yDNA 8.0×10‐8‐1.5×10‐5 5.0×10‐8‐1.2×10‐5 1.0×10‐7‐1.1×10‐5 0.9989 0.9986 0.9981 1.5 3.0 2.5The proposed method had been applied to the determination of the actual samples with satisfactory results. The average recovery was 97.8% and the relative deviations is less than 2.5%. Acknowledgement This work is supported by the Dorctor Foundation of University of Jinan(XBS0901) References Kakehi K, Oda Y, Kinoshita M, Fluorescence polarization: Analysis of Carbohydrate‐Protein interaction, Analytical Biochemistry 2001;297:111–6. Gao F, Luo F, Tang L, Dai L, Wang L. Preparation of a novel fluorescence probe of terbium europium co‐luminescence composite nanoparticles and its application in the determination of proteins. Luminescence, 2008;128(3):462–8. Y. Chen J, Yang JH, Wu X, Wu T, Luan YX. Resonance light scattering technique for the determination of proteins with resorcinol yellow and OP. Talanta, 2002;58:869–74. Chen ZG, Liu GL, Chen MH, Peng YR, Wu MY. Determination of nanograms of proteins based on decreased resonance light scattering of zwitterionic gemini surfactant. Anal. Biochem. 2009;384:337. Tb3+ ‐ protocatechuic acid complex as probe for determination of protein Yanjing Chen and Xiaomei Bi School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Protein is a fundamental material of life and has important roles in physiological processes in all organisms. The quantitative determination of serum albumin, which is the most abundant carrier protein in plasma, is of great importance in biochemistry, biotechnology and immunodiagnostics(1,2). As rare‐earth ions have luminescence characteristics such as narrow spectral width, long luminescence life‐time, large stocks shift and strong binding with biological macro‐molecules, the method would become a valuable tool for determination of proteins using rare‐earth ions and rare‐earth complexes as fluorescence probe(3,4). In this paper, we use protocatechuic acid as ligand for Tb3+ and Tb3+–PCA as a probe for determination of protein. Under the optimum conditions, proteins can increase the fluorescence intensity of the probe and the enhenced intensity is in proportion to the concentration of protein in some range. Based on this, a new fluorescence method for determination of protein is established. To a 10 mL test tube, solutions are added in the following order: PCA,Tb3+, Tris–HCl buffer and BSA. The mixture is diluted to 10 mL with deionized water. After laying aside for 10 min at room temperature, The fuorescence intensity was measured in a 1 cm quartz cell with excitation and emission slits both being 10 nm. The enhenced fluorescence intensity of Tb3+–PCA by BSA at emission peak 546 nm(the exctation wavelength was 320 nm) is represented as ΔF=F0‐F. Here, F and F0 are the intensities of the systems with and without BSA, respectively. The excitation and emission fluorescence spectra of Tb3+, Tb3+‐BSA, Tb3+‐PCA, Tb3+‐PCA‐BSA are shown in Fig. 1(a) and (b). It can be seen that BSA has little effect on the fluorescence spectra of Tb3+, but the BSA can increase the intensity of Tb3+‐PCA significently. Based on the increase of the intensity, the optimum analytical conditions of the system were studied in a series of experiments. The experimental results indicate that ΔF has the largest value in Tris‐HCL buffer at pH8.10 and the optimum volume of buffer is 1.0mL, the optimized concentration is 5.0 × 10−6 mol/L for Tb3+, 6.0 × 10−5 mol/L for PCA, respectively. the addition order of the reagents was selected as follows: PCA‐Tb3+‐BSA‐Tris‐HCl buffer solution. Figure 1. Fluorescence spectra of the system (a) excitation spectra (λem546 nm) (b) emission spectra (λex = 320 nm) 1. Tb3+, 2. Tb3+ ‐ BSA, 3. Tb3+‐PCA, 4. Tb3+‐PCA‐BSA. Conditions: Tb3+ : 5.0 × 10−6mol/l,PCA:6.0 × 10−5 mol/l,BSA: mg/ml, pH = 8.10 Tris‐HCl buffer. A number of foreign substances including metal ions, amino acid and nucleic acids were tested for their effects at BSA concentration 5 μg/mL and the result showed that most substances did not interfere appreciably with the assay. Under the optimum conditions, the linear regression equation was ΔF = 9.569 + 1.761c (µg/mL) and the correlation coefficient was 0.9980. The linear range was from 0.05–30 μg/mL with a detection limit of 0.01 μg/mL. The proposed method had been applied to the determination of the actual samples of human serum albumin with satisfactory results. The recoveries were between 97–104.8%. Compared with the results by the standard method by UV absorption, the relative deviations is less than 4.3%. Acknowledgements This work is supported by the Dorctor Foundation of University of Jinan(XBS0901). References Chen SH, Teixeira J. Structure and fractal dimension of protein–detergent complexes, Phys. Rev. Lett. 1986;57:2583–6. Chen YJ, Yang JH, Wang ZL, Wu X, Wang F. Scopoletine as fluorescence probe for determination of protein. Spectrochimica Acta Part A, 2007;66:686–90. Sun CX, Yang JH, Wu X, Liu SF, Su BY. Study on the fluorescent enhancement effect in terbium–gadolinium–protein–sodium dodecyl benzene sulfonate system and its application on sensitive detection of protein at nanogram level. Biochimie, 2004;86:569–78. Liu RT, Yang JH, Wu X. Study of the interaction between nucleic acid and oxytetracycline–Eu3+ and its analytical application. Journal of Luminescence, 2002;96:201–9. Fluorescence spectroscopic study on the interaction between Bovine Serum Albumin and Silymarin Yanjing Chen and Yuan Guo School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China Silymarin is a plant extract from the seed coat of the silymarin compositaeis known to possess biological activities such as anti‐cardiovascular disease, anti‐tumor and hepatoprotective effect. Serum albumins are plasma proteins contributing significantly to physiological functions and act as carrier proteins. Many drugs are transported in the blood and reach the target tissues by binding to human serum albumin(1,2). There has been practical significance in the study of interactions between ligands and protein. In this paper, the interaction mechanism of silymarin with bovine serum albumin (BSA) in aqueous solution at physiological pH and ionic strength was studied by a fluorescence spectroscopy. When silymarin were added to the BSA solution, a strong fluorescence quenching reaction of silymarin to BSA was observed and based on this, the interaction mechanism was investigated. Into 10 mL colorimetric tube,1.0 mL of BSA solutions (200 μg/mL), Tris‐HCl buffer(pH7.4) and 1.0 mL of 1.5 M NaCl solution were transferred, silymarin solutions of appropriate concentrations were added and diluted to the mark. The samples were excited at 280 nm and the fluorescence spectra was monitored in 300–500 nm using RF‐540 spectrofluorimeter (Shimadzu). The fluorescence intensity data at 345 nm was collected for further study. The excitation and emission slits were both 5 nm, and 1 cm quartz was used. Interaction between BSA and silymarin. The fluorescence spectra of BSA and BSA in the presence of silymarin at various concentrations showed that silymarin can quench the fluorescence emission spectra of BSA and the quenching efficiency increased with the increasing of the concentration of silymarin. The Stern–Volmer relationship was used to analysis the quenching mechanism. The experimental results indicate that the rate constant Kq = 6.82 × 1012 L·mol−1·s−1 for silymarin ‐BSA were greater than 2.0 × 1010 L·mol−1·s−1, the maximum scatter collision quenching constant of quencher to biomacromolecule, and the quenching constant decreases with the temperature increasing. This indicates that the appropriate quenching mechanism of BSA by silymarin is a static quenching process. For static quenching, the following equation was employed to calculate the binding constant K to a site and the number of binding sites n per BSA (3): Figure 1. Overlaping spectra of (1) BSA fluorescence spectra and (2) azorhodanine absorption spectra Conditions: c = cBSA = 3×10‐7 mol·L−1. lg (F0 − F)/ F = lgK + n lg (Q) For the interaction of silymarin to BSA, the calculated result is K = 8.17 × 105 L/mol, n = 1.23(293K); K = 1.79×105 L/mol, n = 1.10(298K). K = 1.40×105 L/mol, n = 1.08(303K). The thermodynamic parameters, enthalpy (ΔH°◦), entropy (ΔS°◦) and free energy change (ΔG°◦), are the main evidence to estimate the binding mode for the complex formation of the small molecule with protein. The acting forces between a small molecule and macromolecule mainly include hydrogen bond, van der Waals force, electrostatic force and hydrophobic interaction force. For the interaction of silymarin to BSA, ΔG = −33.75 kJ/mol(293K), −26.68 kJ/mol(298K), −24.46 kJ/mol(303K); ΔH = −2.134 KJ/mol, ΔS = −238.72 J/mol/K, This indicates that hydrogen bond and van der Waals force play a major role in the interaction of silymarin to BSA. According to the Förster energy transfer theory (4), the shortest binding distance (r) between the acceptor(silymarin) and the donor(BSA) can be obtained from the overlap of the fluorescence spectra of BSA with the absorption spectra of silymarin, we found r = 4.02 nm, far lower than 7 nm (Figure 1). These accord with the conditions of Förster energy transfer theory, and the efficiency of energy transfer E = 0.061. Acknowledgements This work is supported by the Dorctor Foundation of University of Jinan(XBS0901). References Susana S, Nuno M, Victor DF. Interaction of Different Polyphenols with Bovine Serum Albumin (BSA) and Human Salivary α‐Amylase (HSA) by Fluorescence Quenching. J. Agric. Food Chem. 2007;55:6726–35. Jia Z, Yang J, Wu X, Wang F, Guo C, Liu S. Fluorometric determination of proteins using the terbium (III)‐2‐thenoyltrifl uoroacetone‐sodium dodecyl benzene sulfonate‐protein system. Luminescence, 2006;121:535–43. Bian HD, Li M, YU Q, Chen ZF, Tian JN, Liang H. Studyon the interaction of artemisinin with Bovine Serum Albumin, International Journal of Biological Marcromolecules, 2006;39:291–7. Xu JG, Wang ZB. Fluorimetry,(Third Edition) Science Press, Beijing, 2006. First experimental evidence for an intramolecular electron transfer in induced 1,2‐dioxetane decomposition obtained from Hammett linear free‐energy correlations Luiz Francisco ML. Ciscato* and Wilhelm J. Baader Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, SP, 05508‐000, Brazil *E‐mail: ciscato@iq.usp.br The thermal decomposition of spiro‐acridine substituted 1,2‐dioxetanes is one of the few examples of unimolecular dioxetane decomposition resulting in high chemiluminescence emission quantum yields of up to 0.15 E mol−1. Such behavior is in contrast to the known chemiluminescence properties of alkyl‐ or aryl‐substituted 1,2‐dioxetanes, which have very low emission quantum yields of around 1 x 10−4 E mol−1. This strikingly different behavior of the acridine substituted cyclic peroxides may be due to the operation of a different decomposition mechanism, as these derivatives bearing an electron‐donating group. In the case of this kind of 1,2‐dioxetanes, the occurrence of the intramolecular version of the Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism has been postulated to be operating. Accordingly to this mechanism, an electron transfer from the reducing moiety of the molecule to the dioxetanic ring induces the cleavage of the O‐O bond, with the production of a pair of radical ions, whose recombination is able to produce electronically excited singlet state products in high yields. However, although this mechanism is widely accepted to operate in the decomposition of 1,2‐dioxetanes containing electron‐rich substituents and specifically in the induced decomposition of phenolate‐substituted derivatives, there is no experimental evidence of the occurrence of an electron transfer in the initial rate‐limiting step of these transformations. In order to obtain experimental evidence on an electron transfer step in the decomposition of 1,2‐dioxetanes, we studied spiro‐acridine‐substituted 1,‐2 dioxetane (1) derivatives, which contain an aromatic ring bearing substituents with different electronic properties. The rate constants for the thermal decomposition of those 1,2‐dioxetanes show a linear free‐energy correlation for electron withdrawing aromatic substituents, with a Hammett reaction constant of ρ = 1.3 ± 0.1, indicating the development of a partial negative charge in the transition state of the rate‐determining step. Therefore, this result constitutes, for the first time, a direct experimental evidence on the occurrence of an intramolecular electron transfer from the nitrogen atom of the acridine moiety to the 1,2‐dioxetane ring in the rate‐limiting step of the peroxide decomposition. References Baader WJ, Stevani CV, Bastos EL. In The Chemistry of Peroxides; Rappoport, Z., Ed.; wiley: Chichester, U.K., 2006;2(16):1211–78. Lee C, Singer LA. J. Am. Chem. Soc. 1980;102:3823. Nery ALP, Weiss D, Catalani LH, Baader W.J. Tetrahedron 2000;56:5317. Ciscato LFML, Bartoloni FH, Weiss D, Beckert R, Baader WJ. J. Org. Chem. 2010;75:6574. The use of autonomously bioluminescent human cell lines for detection of bacterial contamination Dan Closea,c, Steven Rippb,c, Stacey Pattersona,c and Gary Saylera,b,c aJoint Institute for Biological Sciences, University of Tennessee, Knoxville, TN 37996 bCenter for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996 c490 BioTech, Inc., Knoxville, TN 37996 Although only recently demonstrated to function in mammalian cell lines (1), expression of the fully autonomous bacterial luciferase gene cassette (lux) holds the potential to overcome many of the problems generated through the use of substrate‐dependent bioluminescent reporter systems. With the ability to autonomously produce a visible bioluminescent signal following synergistic expression of its six genetic components, lux cassettes optimized for expression in human host cells can produce detection patterns similar to those of the more common firefly luciferase (luc) system, but without the addition of a chemical luciferin compound (2). Human kidney cells expressing the optimized lux genes were shown to be visible down to population sizes of 1.5 x 104 cells/ml in 24 well tissue culture plates, and as few as 2.5 x 104 cells could be detected through tissue following subcutaneous injection in a nude mouse model (Figure 1). The resulting bioluminescent signal generated from these cells was stable compared to the same cell line expressing a substrate‐dependent luciferase and has been demonstrated to persist across multiple generations of cells without loss of the bioluminescent phenotype (1,2). This stable, autonomous nature of lux bioluminescent production makes it an ideal reporter candidate for the real‐time monitoring of human cell lines. When constitutively bioluminescent cell lines were challenged with bacterial infection, they demonstrated a rapid shift in bioluminescent dynamics (Figure 2). Within 8 hours of infection bioluminescence had significantly decreased compared to uninfected control cells and by 9 hours it had been completely eliminated. While the magnitude of the initial bioluminescent dynamics differed between infection with virulent and non‐virulent strains of E. coli O175:H7, the cessation of bioluminescence resulting from infection of either strain occurred in a similar fashion and across similar timescales. These results demonstrate that the use of autonomous bioluminescence presents a facile method for tracking cellular changes in a remote, automated fashion, without the need for investigator intervention. This makes lux‐based imaging of cell cultures ideal for rapid, high throughput detection of changes in cellular growth and metabolic dynamics while reducing the screening cost compared to traditional substrate‐dependent luciferase systems. Figure 1. The autonomous bioluminescent signal from a) human optimized lux genes is similar in its pseudocolor detection to that of b) human optimized luc gene expression following treatment with its luciferin compound. Figure 2. Human kidney cells expressing human optimized lux genes respond rapidly to infection with E. coli O157:H7. References Close DM, Patterson SS, Ripp S, Baek SJ, Sanseverino J, Sayler GS. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line. PLoS ONE 2010;5(8):e12441. Close DM, Hahn R, Patterson SS, Ripp S, Sayler GS. Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models. J. Biomed. Opt. 2011;16(4):e12441. Chemiluminescence Functionalized Nanoprobes for Bioassays Hua Cui* Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, China E‐mail: hcui@ustc.edu.cn Recently, nanomaterials as biological labels have received increasingly considerable attention in chemiluminescence (CL) and electrochemiluminescence (ECL) bioassays due to their excellent chemical reactivity, catalytic property, surface property, biocompatibility and ease of self‐assembly. Various CL/ECL functionalized nanoprobes have been exploited for bioassays. In these cases, one analytical probe can carry a number of signal reporters so that CL/ECL signals can be greatly amplified. From structural point of view, these CL/ECL functionalized nanoprobes can be divided into two types. One protocol involves the CL reagents indirect capping on the surface of nanomaterials by virtue of bridge molecules. Another protocol is to dope the CL/ECL reagents into nanomaterials. Although these protocols can achieve very high sensitivity for bioassays, there are some drawbacks. For example, analytical process is complicated and time‐consuming; the doped reagents are readily to leak; the labeling procedure is also complicated. These problems limit their practical applications. Thus, it is highly desired to exploit new CL/ECL functionalized nanoprobes with high CL/ECL efficiency, stability and biocompatibility for bioassays. Herein, we report current progress on CL/ECL functionalized nanoprobes for bioassays in our research group. In our group, a direct synthesis strategy was proposed for the preparation of CL functionalized nanoprobes. It was found that CL/ECL reagents, including luminol, isoluminol, N‐(aminobutyl)‐N‐(ethylisoluminol), could directly reduce HAuCl4 or AgNO3 in aqueous solution to form CL/ECL functionalized gold or silver nanoparticles (NPs) 1–4. These CL/ECL functionalized NPs are synthesized via such a simple method and a great number of CL/ECL molecules as stabilizers are coated on the surface of the AuNPs or AgNPs, which exhibited good CL and ECL activities. Subsequently, the CL/ECL functionalized NPs were used as CL/ECL labels to build bio‐probes and ultrasensitive CL/ECL sensors were developed for immunoassays, DNA assays and the detection of small molecules. These bioassays show extremely high sensitivity. Moreover, they are also simple, stable, specific, and time‐saving. Additionally, the labeling procedure is also superior to that of other reported CL/ECL functionalized nanoprobes in simplicity, stability, labeling property and practical applicability. They are of great application potential in the fields of public health, food safety, environmental science and so on. References Cui H, Wang W, Duan CF, Dong YP, Guo JZ. Chem. Eur. J. 2007;13:6975–84. Tian DY, Duan CF, Wang W, Cui* H. Biosens. Bioelectron. 2010;25:2290–5. Chai Y, Tian DY, Wang W, Cui* H. Chem. Commun. 2010;46:7560–2. Tian DY, Zhang HL, Cai Y, Cui* H. Chem. Commun. 2011;47:4959–61. Between emission and perception: do luminous brittle‐stars perceive their own light? Jérôme Delroissea, Jérôme Mallefetb and Patrick Flammanga aLaboratory of marine biology, University of Mons, Belgium; E‐mail: jerome.delroisse@umons.ac.be E‐mail: patrick.flammang@umons.ac.be bLaboratory of marine biology, University of Louvain‐La‐Neuve, Belgium E‐mail: jerome.mallefet@uclouvain.be Keywords: Echinodermata; ophiuroids; bioluminescence; photoreception Since life appeared on earth, light has been one of the most important selective evolutionary forces for living organisms (1). In the marine environment, two predominant phenomena are directly related to light: photoreception and bioluminescence. Bioluminescence is present in at least thirteen phyla and in more than seven hundred identified genera (2). Its implications in the biology of living organisms are multiple (reproduction, nutrition, defense and communication…) (3). In echinoderms, luminescent species predominantly occur in the class Ophiuroidea, the brittle‐stars, which comprises at least 66 species able to emit light (on 175 tested, (2)). In these organisms, luminescence, which is always intrinsic, stems from specialized cells, called photocytes, mainly located along the arms. Recently, molecular markers of photoreception (opsins, arrestin, rhodopsin kinase,…) have been identified in the photophores of the sepiolid squid Euprymna scolopes (4). Bioluminescence in this species is produced by a bacterium ‐ Vibrio fisheri ‐ present in the photophore (extrinsic bioluminescence). Mutant bacteria in which the lux gene is non‐expressed (inducing the lack of bioluminescence) do not persist in this organ (5). These observations suggest that squid photophores would be able to control their own bacterial population though extraocular photosensitivity. Could such a mechanism be present in organisms with intrinsic bioluminescence, such as ophiuroids? One can indeed think that extraocular perception by such organism would constitute an adequate control of photogenesis. The presence of extraocular photosensitivity in a light emitting organ poses some fascinating questions, which have been left unanswered until now. Are luminescent brittle‐stars able to perceive their own light production? Does light detection differ in bioluminescent and non‐bioluminescent species? Do bioluminescent species perceive light in a more efficient way than non‐bioluminescent species, or conversely? These are the questions addressed in this study. A behavioral approach, conducted in aquaria, permitted to highlight the photoreception capabilities of different bioluminescent (blue or green emitters) and non‐bioluminescent brittle‐star species. Depending on the ecology of the targeted brittle‐stars, two different experiments are used. For brittle‐stars considered as relatively photoreactive (Ophiocomina nigra, Ophiopsila aranea…), a high‐intensity illumination is used and the escape behavior is analyzed. For less photoreactive brittle‐stars as for example the borrowing species (Amphiura filiformis, Amphiura chiajei…), a modification of the photoperiod (ambient light) by color restriction is used. A. filiformis is mainly active during the night and is known to use photoreception to perceive the nycthemeral cycles (6). For photoperiod manipulation experiments, light intensity is calibrated using data collected in the field to match natural conditions encountered by the studied species. Specific monochromatic color lighting (red, blue, green, yellow…) are used to target the range of wavelengths these organisms can detect. Different colored LEDs are used for the experiments and their spectra are first evaluated with a microspectrophotometer. The results provide us with new information about the ecology of the luminous brittle‐stars and the putative interaction between the processes of bioluminescence and photoreception. Experiments on the species A. filiformis, a blue light emitter, revealed a spectral sensibility mainly to green light and also to blue light (fig. 1). The photosensibility seems to depend mostly on the ambient light present in the environment (fjord waters at a depth of 30 m where green light is the predominant wavelength), more than on bioluminescence. Work is in progress regarding the non‐burrowing species, the green emitters and the non‐bioluminescent brittle‐stars species. Figure 1. A. Histograms shows average number of “active arms” during the day and night (averaged over 3 days) for five different treatments (green daylight, blue daylight, red daylight, no light, white light. The number of arms is presented on y axis and the time on thex axis. B. Box‐plots oth the data distribution for each treatment during the day and night. Significantly differences are present between the day and the night for the green and the blue daylight treatment. Acknowledgements Work supported by ASSEMBLE (EU contract N°227799). Jérôme Delroisse, Patrick Flammang and Jérôme Mallefet are respectively research fellow, senior research associate and research associate of F.R.S.‐FNRS (Fonds de la Recherche Scientifique). References Land MF and Fernald RD. The evolution of eyes. Ann. Rev. Neuro. 1992;15:1–29. Mallefet J. Echinoderm bioluminescence: where/how and why do so many ophiuroids glow? Bioluminescence in Focus (A collection of illuminating essays) 2009;67–83. Herring PJ. Systematic distribution of bioluminescence in living organisms. J Biolumin Chemilumin. 1987;1(3):147–63. Tong D, Rozas NS, Oakley TH, Mitchell J, Colley NJ, McFall‐Ngai M. Evidence for light perception in a bioluminescent organ. PNAS, 2009;106(24):9836–41. Visick KL, Foster JF, Doino J, McFall‐Ngai M, Ruby EG. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J.Bacteriol 2000;182:4578–86. Rosenberg R, Lundberg L. Photoperiodic activity pattern in the brittle star Amphiura filiformis. Marine Biology 2004;145(4):651–6. Bioluminescent measurement of innate immunity bactericidal factors Dmitrii G. Deryabin and Ilshat F. Karimov Microbiological Department, Orenburg State University, 460018, Orenburg, Russia E‐mail: dgderyabin@yandex.ru Innate immunity comprises molecular and cellular bactericidal mechanisms that protect the host from pathogens in a non‐specific manner. That's why the activity of innate immunity bactericidal factors has important diagnostic and prognostic value, but routine methods of this detection are labour‐consuming and of low technology. The goal of this study is the development of the novel bioluminescent methods for molecular and cellular innate immunity measurement with inherent simplicity, sensitivity, and selectivity. The first group of methods is based on bacteria bioluminescence inhibition that displays a loss of their viability in contact with bactericidal factors. Developed luminescent assay uses recombinant luminescent Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of Photobacterium leiognathi and gives the possibility for differential quantitative determination of blood serum molecular bactericidal systems presented by complement or platelet cationic proteins (PCP). The important step of this procedure is preliminary removal of antibodies from blood serum that excludes influence of specific (adaptive) immunity on the measurement result. The similar principle is used at determination of phagocytosis completeness with neutrophils and macrophages separated after density gradient centrifugation. The luminescent Escherichia coli strain opsonized only by normal human immunoglobulin is used as phagocytosis particles that exclude preliminary loss of its viability in contact with others blood serum components. Developed simultaneous analysis of bacterial destruction and oxygen‐dependent phagocyte system activation also uses chemiluminescent agent luminol and has two variants of realization. The first one is carried out in two separate tests: (i) leukocytes + luminescent bacteria for bioluminescence measurement, (ii) leukocytes + luminol + bacteria with thermoinactivated luminescent system for chemiluminescence measurement. Another variant is carried out in one probe consist of leukocytes, luminol, and luminescent bacteria by means of differentiated measurement of a bioluminescence at ≥ 540 nm and chemiluminescence at ≤ 420 nm. Alternative bioluminescence methods for the differential determination of reactive oxygen species (ROS), including superoxide anion, and hydrogen peroxide, which are formed during phagocytes «oxidative burst» use Escherichia coli strains soxS':: lux and katG ':: lux carrying fusions between promoters of oxidative stress genes and structural luxCDABE genes. The presence of soxS':: lux fusion led to specific bioluminescence induction of bacterial cells treated with N,N′‐dimethyl‐4,4'‐bipyridinium dichloride (paraquat) and katG ':: lux to similar reaction with hydrogen peroxide. Carrying out of same experiments in phagocytosis system led to a primary induction of soxS':: lux fusion at contact with macrophages and katG ':: lux with neutrophils, that can be defined by distinctions in generated ROS spectrum. In addition a luminescence induction of phagocytised bacteria with recA ':: lux fusions it is revealed as SOS‐reaction on DNA damage by ROS and most probably by hydroxyl radical. The developed principles and experimental protocols of bioluminescent analysis of innate immunity bactericidal factors are based on the spectra of bioluminescent viability and gene expression tests and realized on a universal technological platform (figure 1). Figure 1. Principles of bioluminescence innate immunity measurement. Acknowledgments This work was supported by the Russian Foundation of Basic Research grants No. 06‐04‐96906, No. 08‐04‐13726, and No. 11‐04–97064. Fast kinetics of bioluminescent emitting species EV Eremeevaa,b,c, NGH Leferinkc,d, AJWG Visserc, SV Markovaa,b, WJH van Berkelc and ES Vysotskia,b aPhotobiology Lab, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia bSiberian Federal University, Krasnoyarsk 660041, Russia cLaboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands dManchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom Coelenteramide, the product of the photoprotein‐induced bioluminescence reaction, can exist in different ionic forms: a neutral species with a fluorescence emission maximum around 400 nm, the amide mono‐anion (λmax = 450 nm), the phenolate anion (λmax = 480–490 nm), and the pyrazine‐N(4) anion resonance form of the phenolate anion (λmax = 535‐550 nm)1. Based on fluorescence studies of the singlet‐excited state of the phenolate form, it was concluded that the coelenteramide phenolate anion in ion pair contact with a histidine side chain was the light emitter in aequorin and obelin bioluminescence2. Next to the usual emission around 480 nm, photoprotein obelin shows a small emission band around 405 nm, presumably arising from the excited neutral species, and W92F obelin mutant displays an even greater enhancement at 405 nm3,4. From further studies of W92F obelin, it was argued that the bioluminescence of obelin originates from the coelenteramide phenolate ion‐pair excited state with a small admixture of the neutral excited state, both rapidly formed from the primary excited amide anion. In this study the rapid mixing stopped‐flow kinetics of various obelins was measured to study two emitting components with different emission maxima and rates. The rise and decay constants for the violet, originating from the neutral excited state, and blue, from the coelenteramide phenolate ion‐pair excited state, components were calculated. The rates of bioluminescence rise of W92F obelin were shown to be moderately influenced by temperature. The implications of these results for the photoprotein bioluminescence mechanism are discussed. Supported by the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058) and Wageningen University Sandwich PhD‐Fellowship program. References Shimomura O, Teranishi K. Light‐emitters involved in the luminescence of coelenterazine. Luminescence. 2000;15:51–8. Mori K, Maki S, Niwa H, Ikeda H, Hirano T. Real light emitter in the bioluminescence of the calcium‐activated photoproteins aequorin and obelin: light emission from the singlet‐excited state of coelenteramide phenolate anion in a contact ion pair. Tetrahedron 2006;62:6272–88. Markova SV, Vysotski ES, Lee J. Obelin hyperexpression and characterization. Bioluminescence and Chemiluminescence, World Scientific, Singapore, 2001;115–8. Deng L, Vysotski ES, Liu ZJ, Markova SV, Malikova NP, Lee J, Rose J, Wang BC. Structural basis for the emission of violet bioluminescence from a W92F obelin mutant. FEBS Lett. 2001;506: 281–5. Gelatin and starch as stabilizers of bacterial luciferase and oxidoreductase Anna Bezrukikha, Elena Esimbekovab,a and Valentina Kratasyuka,b aSiberian Federal University, Krasnoyarsk, 660041, Russia, E‐mail: aebezrukih@gmail.com bInstitute of Biophysics, Krasnoyarsk, 660036, Russia An immobilized reagent for bioluminescent analysis, stable during storage and usage, is currently being developed (1) on the basis of a coupled enzymatic system of luminous bacteria NADH:FMN‐oxidoreductase‐luciferase. The aim of this work was to evaluate gelatin and starch as agents for increasing the thermal stability of bioluminescent enzymes. Using buffer solution as a control, we studied the activity level and thermal inactivation of the coupled enzymatic system and, separately, oxidoreductase in the presence of gelatin and starch. It was shown that the activity of the coupled enzymatic system and the kinetics of their thermal inactivation were different in gelatin that did not form a gel structure (sol) and in gel. The enzymes decreased their bioluminescence intensity in gelatin sol, but the inclusion of enzymes into a gel matrix resulted in their stabilization and greater activity. The presence of starch did not cause any considerable change in enzyme activity, compared with the control values. The temperature optimum of the coupled enzymatic system was 33 °C in the presence of starch, a significantly higher temperature in comparison with the control. However, in the presence of gelatin, its temperature optimum was lower due to its gelatinization. The thermal inactivation of the coupled enzymatic system involved two stages of enzymes inactivation: dissociation of the enzymes on subunits, followed by denaturation (2). Analysis of the effective thermal inactivation rate constants (k1ef and k2ef) revealed that gelatin accelerated the first stage of thermal inactivation, while starch reduced the second one. For example, the effective thermal inactivation rate constant at 43 °C was decreased 12 times in the presence of starch. The kinetics of oxidoreductase and the coupled enzymatic system thermal inactivation in gelatin were indistinguishable. Changes of oxidoreductase thermal inactivation rates didn't take place in the presence of starch (Table 1). Table 1. Effective rate constants of the first (k1ef) and the second (k2ef) thermal inactivation stages of NADH:FMN‐oxidoreductase in the presence of 1% gelatin, 2% starch and buffer solution (control) at different temperatures T, °C Buffer Gelatin Starch k1ef×102, min‐1 k2ef×102, min‐1 k1ef×102, min‐1 k2ef×102, min‐1 k1ef×102, min‐1 k2ef×102, min‐1 25 11±2 0,7±0,1 19±3 1,0±0,2 14±2 0,53±0,08 30 35±5 1,4±0,2 40±6 0±0,05 36±5 1,6±0,2 35 60±9 0,8±0,1 86±13 0,39±0,06 59±9 0±0,05 40 87±13 1,3±0,2 104±16 1,5±0,2 93±14 1,2±0,2 Both gelatin and starch have some stabilizing effect on the enzymes of bioluminescent bacteria. However, starch is the better additive for increasing the thermal stability of the coupled enzymatic system of luminous bacteria. This work was supported by the Federal Agency of Science and Innovations (contract No 02.740.11.0766), Russian Academy of Sciences (Program “Molecular and cellular biology”, grant No 6.2), President of RF (grant Leading scientific school No 64987.2010.4), Program of the Government of the Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). References Esimbekova EN, Torgashina IG, Kratasyuk VA. Disk‐shaped immobilized multicomponent reagent for bioluminescent analyses: correlation between activity and composition. Enzyme and microbial technology 2007;40(2):343–6. Bezrukikh AE, Esimbekova EN, Kratasyuk VA. Thermal inactivation of the coupled enzymatic system of luminous bacteria NADH:FMN‐oxidoreductase‐luciferase in gelatin. Journal of Siberian Federal University. Biology 2011;4:64–74. Recent advances in the theoretical research of the firefly multicolor bioluminescence Luís Pinto da Silva and Joaquim C.G. Esteves da Silva* Centro de Investigação em Química (CIQ‐UP), Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre 687, 4169‐007 Porto (Portugal). Firefly luciferase catalyzes the oxidation of firefly luciferin, giving rise to light in a two‐step reaction.1 The most relevant characteristics of this system are: the formation of an excited state emitter (oxyluciferin), its high quantum yield and its pH‐dependent multicolor bioluminescence.1,2 These features have encouraged many researchers to develop several applications for this system.3 Computational methodologies have been fundamental in the precise definition of the elusive color tuning mechanism. The dissociation and tautomeric reactions involved in the chemical equilibrium of oxyluciferin were examined computationally, both in the ground and in the excited state.2 For establishing a more realistic model, implicit solvation was included in the calculations in order to simulate different degrees of polarity. Having defined the anionic keto‐form species as the light emitter (Figure 1), the interaction of this molecule with several small molecules was studied. Theoretical calculations demonstrated that the color of light emitted by the bioluminophore can be modulated by modification of the intermolecular interactions formed between the light emitter and other molecules.4,5 Computational methodologies were also employed in the study of the interaction between Luciola cruciata luciferase and excited state anionic oxyluciferin.5,6 It was demonstrated that the red‐shift verified in this system results mainly from decreased interaction of oxyluciferin with AMP and increased interaction with Phe249. The rearrangement of the hydrogen‐bond network is also instrumental. Figure 1. Representation of anionic keto‐form oxyluciferin as the solo light emitter. References Marques SM, Esteves da Silva JCG. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions. IUBMB Life 2009;61:6–17. Pinto da Silva L, Esteves da Silva JCG. Computational Studies of the Luciferase Light‐Emitting Product: Oxyluciferin. J. Chem. Theory Comput. 2011;7:808–17. Roda A, Guardigli M. Analytical chemiluminescence and bioluminescence: latest achievements and new horizons. Anal. Bioanal. Chem. 2011; DOI: 10.1007/s00216‐011‐5455‐8. Pinto da Silva L, Esteves da Silva JCG. Computational investigation of the effect of pH on the color of the firefly bioluminescence by DFT. Chemphyschem 2011;12:951–60. Pinto da Silva L, Esteves da Silva JCG. Theoretical modulation of the color of light emitted by firefly oxyluciferin. J. Comput. Chem. 2011;32:2654–63. Pinto da Silva L, Esteves da Silva JCG. Study of the Effects of Intermolecular Interactions on Firefly Multicolor Bioluminescence. Chemphyschem 2011;12:3002–8. A firefly luciferase‐based turn‐on sensor for biothiols Danielle M. Fontaine*, Jessica Yi and Bruce R. Branchini Department of Chemistry, Connecticut College, New London, CT 06320, USA E‐mail: brbra@conncoll.edu Biological thiols like cysteine (Cys), homocysteine (Hcy) and reduced glutathione (GSH) are important markers for the diagnosis of many metabolic disturbances and human disease states (1). Convenient, sensitive and specific assays for biothiols in human plasma are therefore of considerable interest in clinical chemistry. Our aim is to develop a firefly luciferase‐based turn‐on biosensor for biothiols building on previous studies (2) with luciferase variants containing surface Cys residues that were intramolecularly cross‐linked with bifunctional maleimide reagents. In this initial study, we produced encouraging proof‐of concept results in which 1 mM N‐acetyl cysteine (NAC) elicited an ~85–fold increase in the dim bioluminescence produced initially by a novel disulfide cross‐linked luciferase. Based on molecular modeling results with P. pyralis and having constructed (2) the variant Ppy 9−, a stabilized luciferase lacking Cys residues, we designed and produced Ppy 10− C290/C476, a red light‐emitting enzyme containing surface Cys residues at the N‐domain position 290 and C‐domain position 476. Upon gentle oxidative treatment with 50 μM ferricyanide for 10 d at 5 °C, we intramolecularly cross‐linked the N‐ and C‐domains through disulfide bond formation, which was confirmed by Ellman's (sulfhydryl) assay, LC/ESI‐MS and SDS‐PAGE. An ~100‐fold loss of specific activity measured with luciferin and Mg‐ATP was observed as a result of the disulfide trapping. As anticipated, the addition of thiol NAC (1 mM), DTT (20 mM) or TCEP (50 mM) to solutions of the cross‐linked enzyme results in the restoration of 80–90% of the original (uncross‐linked) bioluminescence activity of Ppy 10− C290/C476. The reaction of NAC at 20 °C and pH 8.6 presumably represents attack of the thiolate anion that results in the breaking of the disulfide linkage, producing free, active enzyme. This result formed the basis for this assay of biothiols that, likewise, are capable of liberating active enzyme by reaction at the disulfide cross‐linked site. For example, using 5 µM solutions of the cross‐linked luciferase, we have detected 20 µM Cys, 10 µM NAC and 10 µM GSH; while 50 µM His and Met did not produce signals above background under the same assay conditions. We will present our results on the determination of the dynamic ranges and detection limits for Cys, Hcy, GSH, the common α‐amino acids, and various other plasma constituents. Additionally, results will be presented using a near‐Infrared emitting version of the disulfide cross‐linked luciferase. References Packer L, Cadenas E. Biothiols in health and disease, M. Dekker, New York, 1995. Branchini BR, Rosenberg JC, Fontaine DM, Southworth TL, Behney CE, Uzasci L. Bioluminescence Is Produced from a Trapped Firefly Luciferase Conformation Predicted by the Domain Alternation Mechanism, JACS 2011;133:11088–91. Bioluminescent re‐engineered proteins as effective reporters for in vitro assay LA Franka,b, VV Krasitskayaa, AN Kudryavtseva,b, LP Burakovaa,b, GA Stepanyuka, SV Markovaa and ES Vysotskia aPhotobiology Laboratory, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia bSiberian Federal University, Krasnoyarsk 660041, Russia Nowadays, the light‐emitting proteins are promising analytical tool both for in vitro and in vivo application to satisfy growing demands of science and medicine. Majority of the analytical techniques developed in the last ten years are based on coelenterazine‐depended bioluminescent systems – Ca2+‐regulated photoproteins and luciferases. Binding assay based on these bioluminescent reporters provides high sensitivity, robustness, reproducibility and safety (1). Attempts to vary the proteins properties (e.g. to improve stability, to alter their bioluminescence spectral characteristics, kinetics, etc.) using site‐directed mutagenesis were undertaken since cDNAs of the proteins have been cloned. Re‐engineered proteins essentially broaden bioluminescence‐based assay facilities. The recent trend in analytical investigations includes development of multianalyte bioluminescence‐based assay. Using site‐directed mutagenesis a number of proteins with unique bioluminescence were obtained. Obelin mutants with essentially altered bioluminescence spectral and kinetic characteristics were obtained in our lab: W92F‐H22E emitting fast (kd = 0.6 s−1) violet signal (λmax = 387 nm) and Y138F with slow (kd = 6.1 s−1) greenish light (λmax = 498 nm). At that, overlap of the bioluminescence spectra is minor (2). Applying those as reporters, we developed a dual‐analyte single‐well bioluminescence assay. Next we used it for simultaneous immunoassay of two gonadotropic hormones, of two prolactin forms or for detection of two alleles at SNP genotyping. One more re‐engineering type is genetic fusing of light‐emitting protein with the proteins of interest. Obtained bifunctional molecules contain bio‐specific and reporter modules and are effective markers for in vivo and in vitro assays. The main advantages of the labels obtained as fused proteins are: (1) the lack of bulky chemical cross‐linking stages, (2) high activity of enzyme module, not subjected to any chemical treatments and (3) usually simple way to purify chimeric proteins with affinity chromatography. Obelin and luciferase Renilla muelleri were genetically fused with several bio‐specific molecules and the bioluminescent and analytical properties of these chimeras were examined. References Frank LA. Sensors 2010;10:11287–300. Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES. Anal. Bioanal. Chem. 2008;391:2891–6. Acknowledgments This work was supported by grant No.76 of the RAS SB, and by the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058) Study on the second order scattering spectrum of new type rhodanine derivative‐neomycin in micromulsion Shenguang Ge, Xiuling Jiao* and Dairong Chen* School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, People's Republic of China; E‐mail: Dairong Chen, E‐mail: jiaoxl@sdu.edu.cn; cdr@sdu.edu.cn Neomycin (NEO) was one of aminoglycoside antibiotics which were used widely in the clinic, agriculture, animal husbandry, fisheries industry(1). Hence, it was very important to monitor the aminoglycoside antibiotics to protect human health. Second order scattering (SOS) is a high sensitive mentod and it would become a new analysis method (2). In this work, 3‐(4'‐methylphenyl)‐5‐ (2'‐sulfophenylazo) rhodanine (4MRASP) was synthesized by the author. The SOS spectra of 4MRASP‐SDS‐NEO (SDS: sodium dodecyl sulfate) system and its blank were obtained by fixing λex = 1/2 λem at the range of 220 ~ 420 nm, respectively (Figure 1). It could be seen that, under the experimental condition, SOS wavelength of system didn't change obviously when NEO was added, but the light scattering intensity was enhanced rapidly, which was caused by the formation of 4MRASP‐SDS‐NEO ion‐association complex. Also it could be found fom Figure 1 that the maximal SOS peak was at λex/λem = 310 nm/620 nm, which was chosen for the determination of NEO. Figure 1. Wavelength scaning of SOS 1. reagent black. 1′. reaction product. Under the experimental condition, the SOS spectra were scanned (Figure 2). It could be seen from Figure 2 that the SOS intensities of the 4MRASP and NEO were weak, 4MRASP+SDS and 4MRASP+NEO also had weak SOS intensities, which indicated a very weak interaction between 4MRASP and SDS or between 4MRASP and NEO. However, when 4MRASP, SDS and NEO were mixed together, the SOS intensity of the system was enhanced rapidly. It could be concluded that the interaction among three molecules had occurred and made three molecules form a new complex which enhanced SOS signals. It could be also found that the SOS intensity of the system increased obviously with increasing concentration of NEO. Based on this, a new determination method of NEO was established. Figure 2. SOS spectrums of different systems (λex/λem = 310 nm/620 nm) 1. 4MRASP+SDS; 2. 4MRASP; 3. NEO; 4. 4MRASP+NEO; 5. 4MRASP +NEO(0.2 mL)+SDS; 6. 4MRASP+NEO(0.3 mL) + SDS. pH 1.94. The intensity of the SOS was proportional to the concentration of NEO in the range of 0 ~ 1.6 µg·mL‐1, based on this, a novel determination method of NEO at nanogram level with high sensitivity and good selectivity had been developed. The interaction among molecules was studied in detail with quantum chemistry, thermodynamics and spectroscopy. The possible mechanism of sensitization effect of SDS was proposed. Acknowledgements This work was financially supported by Natural Science Foundation of Shandong Province, China (ZR2011BQ019), Natural Science Research Foundation of China (21175058) and Technology Development Plan of Shandong Province, China (Grant No. 2011GGB01153). References Liang X, Hongjuan X, Sunil K, David G, Erik D, Paris H, Michael S, Dev PA. Probing the Recognition Surface of a DNA Triplex: Binding Studies with Intercalator−Neomycin Conjugates. Biochem. 2010;49(26):5540–52. Ding F, Zhao HC, Chen SL, OuYang J, Jin LP. Study of the interaction of nucleic acid with europium(III) and CTMAB and determination of nucleic acids at nanogram levels by the second‐order scattering. Anal. Chim. Acta. 2005;536:171–8. Catalytic fluorescence method for determination of trace vanadium Shenguang Ge, Xiuling Jiao and Dairong Chen* School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, People's Republic of China; *Corresponding author: Dairong Chen, E‐mail: cdr@sdu.edu.cn Vanadium was extensively applied in industry, the analysis of trace vanadium was more and more important. In present, there was different methods of determination vanadium: for example, spectrophotometry, catalytic spectrophotometry, atomic absorption spectrometry, ICP‐AES (1–4). In recent years, catalytic dynamic spectrofluorimetry is becoming a powerful determination method because of its high sensitivity, precision and simple operation. Figure 1. Excitation spectra and emission spectra 1,1′ 4MRAAP‐KIO4‐V; 2,2′ 4MRAAPKIO4; 3,3′ 4MRAAP. The method was based on the catalytic effect of vanadium on oxidation of 3‐(4'‐methylphenyl)‐5‐(2'‐arsenoxylphenylazo)rhodanine (4MRAAP) by potassium periodate in potassium hydrogen phthalate‐sodium hydroxide buffer solution (pH = 5.2). It can be found in the excitation spectra and emission spectra (Figure 1) that the fluorescence intensity of 4MRAAP itself is weak (curve 3, 3'), but we can see from the graph that the fluorescence intensity of the catalyzed system 4MRAAP‐KIO4‐V (curve 1,1') is evidently enhanced than the uncatalyzed system 4MRAAP‐KIO4 (curve 2, 2'), which is produced the catalytic action of trace vanadium. 309 nm and 406 nm have been chosen to be the wavelength of excitation peak and emission peak at which determination for trace vanadium by 4MRAAP was carried out. Under the optimum conditions, the apparent activation energy of this reaction was 52.81 kJ/mol, the reaction rate constant was 0.34/s, the relative fluorescence intensity has a linear relationship against the concentration of vanadium in the range of 0.10 ~ 10.0 µg/L with a regression equation of ΔI = 10.97+73.73ρ (µg/L) and correlation coefficient (r) of 0.9934. The detection limit of vanadium was found to be 2.8 × 10−8 g/L. The method can be used to determine trace amount of vanadium in real samples. The R.S.D. of method was less than 2.56%. The recovery was between 97.2% and 104.1%. Keywords: vanadium; catalytic fluorescence method; 3‐(4'‐methylphenyl)‐5‐(2'‐ arsenoxylphenylazo) rhodanine Acknowledgements This work was financially supported by Natural Science Foundation of Shandong Province, China (ZR2011BQ019), Natural Science Research Foundation of China (21175058) and Technology Development Plan of Shandong Province, China (Grant No. 2011GGB01153). References Leila Rostampour, Mohammad Ali Taher. Determination of trace amounts of vanadium by UV–vis spectrophotometric after separation and preconcentration with modified natural clinoptilolite as a new sorbent. Talanta 2008;75(5):1279–83. Graç M, Korn A, Santos DSS, Welz B. Atomic spectrometric methods for the determination of metals and metalloids in automotive fuels – A review. Talanta 2007;73:1–11. Khuhawar MY, Arain GM. Liquid chromatographic determination of vanadium in petroleum oils and mineral ore samples using 2‐acetylpyridne‐4‐phenyl‐3‐ thiosemicarbazone as derivatizing reagent. Talanta 2006;68(3):535–41. Alisa Rudnitskaya, Dmitry V. Evtuguin, Jose AF. Gamelas, Andrey Legin. Multisensor system for determination of polyoxometalates containing vanadium at its different oxidation states. Talanta 2007;72(2):497–505. Chemiluminescence of higher fungi J Gitelson, V Bondar, E Rodicheva, S Medvedeva and G Vydryakova Institute of Biophysics (Russian Academy of Sciences, Siberian Branch), Institute of Fundamental Biology and Biotechnology (Siberian Federal University) E‐mail: jigit@rogers.com, gitelson@ibp.ru Chemiluminescence was studied in species of higher fungi having no visible bioluminescence. Measurements were made in Siberia in summer of 2011. The objects under study were 150 samples of higher fungi collected in the forests of Krasnoyarsk Territory in the coastal zone of the Yenissei river, in the neighborhood of Krasnoyarsk city in latitude 56°02'N and longitude 93°04'E. Light emission was studied on samples taken from different parts of the fruiting body. Each samples weighed in air‐dry condition to calculate specific emition per unit mass. Measurements have been made with Glomax 20/20 «Promega» luminometer (USA). Measurements of each sample were recorded for 10 seconds. All species of studied higher fungi have been found to have chemiluminescent radiance. Visually their glow is not detected by unaided eye adapted to darkness, i.e. they are to be attributed to non‐bioluminescent. Different species exhibit different chemiluminescence intensity which varies from 2.51 x 105 to 2.22 x 108 quantum x sec−1 x g−1, i.e. in 1000 times. Fruiting body of picked up fungi maintains its emissivity for dozens of hours and is discernibly depressed by dehydration. Mechanical damage of the tissue brings forth a radiant flash with specific decay kinetics. Comparison of the species composition allows to conclude that the chemiluminescence is the strongest in Russulales and Agaricales, much weaker in Boletales and Polyporales fungi growing on tree trunks. Among the studied fungi the highest intensity is exhibited by representatives of Russula foetens (3.31 x 107–2.22 x 108 quantum x sec−1 x g−1) and Russula ochroleuca (up to 1.27 x 108 quantum x sec−1 x g−1). The above data make possible to state that the chemiluminescent emission is intrinsic for the fruiting body tissues of many fungi species. We did not study mycelium from other fungus organs in this respect, except for Armillaria borealis, luminescence of its mycelium is registred. In literature it is well known the bioluminescence of other Armillaria species: A. gallica, A. mellea (1). The existence of a weak glow in bioluminescent fungi was first noted by O. Shimomura (2). Weak radiance of mycelium in some representatives of Basidiomycota, Ascomycota and Zygomycota was described by Michail J.D and Bruhn J.N (3). As a hypothesis calling for further investigations we suggest that the described fungal chemiluminescence is the metabolic basis where the evolution intensified this function to give rise to fungal bioluminescence. Our immediate task for fungal chemiluminescence research is to define metabolic source of their emission. The authors thank N.P. Kutafeeva for help in determining the taxonomic position of studied fungi. The work has been done with partial support by the Federal Agency for Science and Innovation within the Federal Special Purpose Program (contract № 02.740.11.0766) and the Program of the Government of Russian Federation «Measures to Attract Leading Scientists to Russian Educational Institutions» (grant No 11. G34.31.058) References Desjardin DE, Oliveira AG, Stevani CV. Fungi bioluminescence revisited. Photochem. Protobiol. Sci. 2008;7:170–82. Shimomura O. Bioluminescence: chemical principles and methods. World Scientific Publishing Co. Pte. Ltd. 2006;470 Michail JD and Bruhn JN. Bioluminescence is widespread within the kingdom of fungi. Opera Mycologica, 2007;1:28–33. First description of neural control mechanisms in bioluminescence of Tomopteris helgolandica (Annelida, Polychaeta) A Gouveneaux and J Mallefet Catholic University of Louvain, Marine Biology Laboratory, Place Croix du Sud, 3, bt L7.06.04, Louvain‐la‐Neuve, 1348, Belgium Gouveneaux A. is FNRS FRIA PhD student, Mallefet J. is research associate of FNRS Transparency is a common passive cryptic adaptation in pelagic environment.(1) In fact, by reducing light reflection and light scattering, biological transparency makes organism invisible. Paradoxally, many gelatinous zooplankton species are able to produce visible light.(2) So, what ecological advantage(s) bioluminescence could provide to such organisms? The model studied here is Tomopteris helgolandica, a transparent planktonic worm collected by trawling between 250 and 300 m depth in fjords near Bergen (Norway). The specimens were picked out and maintained during few days in sea water containers placed in a dark cold room. The bioluminescence potential was measured on pieces of three pairs of parapods from anaesthetized organisms. Each preparation was stocked in cold artificial sea water up to chemical induction of bioluminescence response. Available data about tomopterids bioluminescence were limited to anecdotic observations and morphological descriptions, poorly documented.(3) In way of having a global view of its bioluminescent potential, different aspects of the light emission process are currently explored, including neural basis of physiological control. After a screening of major pharmacological components, investigation has been oriented in cholinergic control hypothesis. It has been confirmed by dose‐dependent emission of light in response to carbachol stimulation (Fig. 1). Inhibitory effect of tubocurarine on light emission suggested that nicotinic receptors were involved in the signal transmission pathway leading to light emission (Fig. 2). These results constitute the first data about neural control mechanisms in bioluminescence of a pelagic worm. Two annelid families have been previously studied and both concerned benthic species.(4) Only pharmacological investigations have been conducted so far but these results will be completed by electrophysiological and immunohistochemical approaches. References Johnsen S. Hidden in Plain Sight; The Ecology and Phyisiology of Osganismal Transparency, Biol. Bull. 2001;201:301–18. Haddock SHD, Case JF. Bioluminescence spectra of shallow and deep‐sea gelatinous zooplankton: ctenophores, medusa and siphonophores, Mar. Biol. 1999;133:571–82. Dales RP. Bioluminescence in Pelagic Polychaetes, J. Fish. Res. Bd. Canada 1971;28:1487–89. Anctil M. Neural control mechanisms in bioluminescence, NATO ASI series. Series A: life sciences 1987;141:573–602. Figure 1. Effect of carbachol concentration on luminescence of parapods preparations from Tomopteris helgolandica. Intensities of light emitted are expressed as a percentage of those measured in control preparation, stimulated by 200 mM KCl solution Mean ± SEM, n = 6, * indicates a difference between this response and the higher carbachol‐induced one (10−3 M) (P < 1,05). Figure 2. Inhibitory effect of tubocurarine on carbachol‐induced luminescence of parapods preparations from Tomopteris helgolandica. Intensities of light emitted are expressed as a percentage of those measured in control preparations not treated with cholinergic antagonists Mean ± SEM, n = 6, * P = 0,0553. Chemiluminescence measurement of autotaxin activity in human serum M Guardiglia, M Di Fuscob, M Mirasolia, P Simonic, F Azzarolic, G Mazzellac and A Rodaa aDepartment of Pharmaceutical Sciences, University of Bologna, Bologna, Italy bAdvanced Applications in Mechanical Engineering and Materials Technology Interdepartmental Center for Industrial Research, University of Bologna, Bologna, Italy cDepartment of Internal Medicine, University of Bologna, Bologna, Italy E‐mail: aldo.roda@unibo.it Autotaxin (ATX) is an extracellular lysophospholipase D that converts lysophosphatidylcholine (LPC) into the lipid signalling molecule lysophosphatidic acid (LPA). Recent experimental evidence suggested that ATX and LPA are potential mediators of cholestatic pruritus (1), since ATX activity was found to be increased in patients with cholestatic disorders but not in other forms of pruritus. Furthermore, ATX levels strongly correlated with therapy efficacy in pruritic cholestatic patients. A number of different analytical methods have been employed to measure ATX levels (2), based either on the detection of the molecule by immunoassays or on the evaluation of its enzymatic activity using radiolabeled or fluorogenic substrates. The most common approach is the photometric one, in which the choline liberated from the hydrolysis of LPC is detected using a dual‐enzymatic assay employing choline oxidase and horseradish peroxidase in the presence of a chromogenic peroxidase substrate. We have developed a chemiluminescent (CL) assay for the measurement of ATX activity exploiting the bis‐2,4,6‐(trichlorophenyl)oxalate (TCPO) CL reaction for detecting hydrogen peroxide produced by a series of coupled enzyme reactions involving ATX in the presence of exogenously added LPC (Figure 1). A similar approach has been previously employed for the evaluation of recombinant urate oxidase (Rasburicase) activity in serum (3). Preliminary results showed that ATX could be measured at low concentrations (LOD < 1 µg/L) using shorter incubation steps (1–2 h) with respect to colorimetric methods. Assay of ATX activity in serum required 1:100 or 1:200 (v/v) sample dilution to avoid matrix effect. Nevertheless, the limit of detection in serum (LOD < 0.1 mg/L) was still suitable for accurate measurement of physiological ATX levels (about 1 mg/L) and the evaluation of its overexpression. In comparison to other reported methods, this one is fast and reliable and could therefore represent a useful tool for the diagnosis of cholestatic pruritus. Thanks to the high sensitivity of CL detection, this assay could be also employed in miniaturized analytical devices. Figure 1. Scheme of the reactions involved in the CL ATX activity assay. References Kremer AE, et al. Lysophosphatidic acid is a potential mediator of cholestatic pruritus. Gastroenterology 2010;139:1008–18. Tokumura A. Bpa Mol Cell Biol Lipids 2002;1582:18. Sestigiani E, et al. Efficacy and (pharmaco)kinetics of one single dose of rasburicase in patients with chronic kidney disease. Nephron Clin Pract 2008;108:c265–71. Estimation of hydrodynamic volumes of NADH and FMN molecules in viscous media by fluorescence anisotropy technique DV Gulnova, EV Nemtsevaa,b, MA Gerasimovaa and VA Kratasyuka,b aSiberian Federal University, 79 Svobodny Prospect, Krasnoyarsk, 660041, Russia bLab. of Photobiology, Institute of Biophysics SB RAS, 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia Introduction Viscous media can be considered as the simplest models of intracellular environment for the enzymes. To reveal the peculiarity of enzymes functioning in cytoplasm the mechanisms of bacterial bioluminescent reaction in viscous media have being investigated. Earlier it was found that the presence of glycerol and sucrose reduces the intensity of bacterial bioluminescence reaction (1). One of the possible mechanisms is the modification of substrate‐enzyme interactions in the presence of co‐solvent due to steric hindrance. The aim of this work was to estimate hydrodynamic volumes of main substrates of bioluminescent reaction in bacteria – FMN and NADH in viscous media from fluorescence anisotropy data. Experimental The following reagents were used: FMN (Serva), NADH (Gerbu), glycerol (Gerbu), sucrose (Gerbu). Emission, excitation spectra and anisotropy of fluorescence were recorded with luminescent spectrometer Aminco Bowman Series 2 (Thermo Spectronic, the USA). Fluorescence lifetimes of NADH were measured with spectrofluorimeter Fluorolog 3‐22 (Horiba Jobin Yvon, France) equipped with TCSPC. Measurements were performed at 20°C. Hydrodynamic volumes were calculated using the Perrin and Stokes‐Einstein equations fusion: r0r=1+kBTηVτ, (1) where r0 is the fundamental anisotropy, r is the measured anisotropy, kB is the Boltzmann constant, T is the temperature, τ is the fluorescence lifetime, η is the viscosity, V is the hydrodynamic volume of the fluorescent molecule (2). It should be noticed that equation (1) assumes spherical shape of emitting molecule. Results and discussion Photophysical characteristics of FMN in media containing glycerol, sucrose, starch and gelatin, was investigated previously (3). Increase of fluorescence lifetime and anisotropy was found, while spectral shifts were not. The analogous study of NADH revealed increase in fluorescence lifetime from 0,35 ns in buffer to 0,57 ns and 1,8 ns at the presence of glycerol and sucrose, respectively. The pronounced blue shift of NADH emission maximum, up to 20 nm, was observed in the media with increasing viscosity probably due to slowdown of solvent relaxation. The fluorescence anisotropy rise was measured (Table 1, rexp). Whole data set allowed estimation of hydrodynamic volumes of studied nucleotide molecules in the presence of glycerol and sucrose. The equation (1) was used for calculation, r0 was taken as 0,354 and 0,37 for FMN and NADH respectively. The results are shown in the Table 1. Table 1. Estimated hydrodynamic volumes V of the FMN and NADH Nucleotide Cosolvent C, % η, cP τ, ns rexp V, 10‐21 cm3 FMN Buffer 0 1,0 4,69 0,0078 0,429 FMN Glycerol 10 1,31 4,75 0,0094 0,399 30 2,50 4,86 0,0177 0,369 50 6,05 4,95 0,0241 0,220 70 22,94 4,96 0,0568 0,167 FMN Sucrose 10 1,35 4,72 0,0725 3,64 30 3,19 4,81 0,07454 1,63 50 15,43 4,95 0,07628 0,356 NADH Buffer 0 1,0 0,35 0,1088 0,589 NADH Glycerol 20 1,77 0,38 0,1184 0,409 40 3,75 0,45 0,1108 0,207 60 10,96 0,50 0,147 0,122 80 62,00 0,57 0,1375 0,022 NADH Sucrose 10 1,35 1,04 0,1626 2,44 20 1,95 1,49 0,238 5,59 40 6,17 1,80 0,2218 1,77 It was found that increase in anisotropy value could not be explained only by higher viscosity and fluorescence lifetimes change. The apparent volumes of the nucleotides are also varied in viscous solutions. Thus, the study has confirmed that some steric hindrance can appear during interaction of enzymes with FMN and NADH in media containing glycerol and sucrose. References Sukovataya IE, Kaykova EV, Buka NS, Zadorozhnaya L. Kinetics of bacterial coupled enzymatic system NAD(P)H:FMN‐oxidoreductase‐luciferase catalysis in solvents of increased viscosity. Luminescence 2008;23(2):93. Lakowicz JR. Principles of Fluorescence Spectroscopy, Springer, New York, 2006. Nemtseva EV, Gulnov DV, Gerasimova MA. Photophysical characteristics of flavinmononucleotide in viscous media. Luminescence 2010;25(2):191–2. Diversity and evolution of calcium‐activated photoproteins Steven HD. Haddock, Meghan L. Powers, Nathan C. Shaner, Amy G. McDermott and Lynne M. Christianson Monterey Bay Aquarium Research Institute, 7700 Sandholdt Rd., Moss Landing, CA 95039 A variety of organisms use calcium‐activated photoproteins in conjunction with the luciferin coelenterazine to make light. Although the organisms are spread across at least three different phyla, and several classes within those phyla, the corresponding genetic sequences of the photoproteins show several conserved motifs, and the proteins are clearly homologous. Photoprotein genes have been cloned from subclass Hydroidolina of the Hydroza and ctenophores, and other similar gene sequences have been found by searching the genomes of a non‐luminous anthozoan and even a sponge. Other taxa have homologous photoproteins which have only been characterized chemically, or not at all. These include radiolarians (both phaeodarian and polycystine types), trachyline hydromedusae, and siphonphores. We have targeted these taxa as well as additional ctenophores for transcriptome sequencing. We will present a phylogenetic tree of known photoproteins along with other calcium‐binding proteins, and discuss the evolution of this important category of bioluminescent molecules. Color‐tuning in photo‐functional proteins: electronic structure and interactions Jun‐ya Hasegawa Fukui Institute for Fundamental Chemistry, Kyoto University; 34‐4 Takano‐Nishihiraki‐cho, Sakyo‐ku, Kyoto 606‐8103, Japan In vision and fluorescent proteins, controlling photo‐absorption/emission energy of chromophore is essential to furnish a protein with the photo‐functionality. Retinal Schiff base in vision, luciferin in insects, and green‐fluorescent protein chromophores in a jellyfish are the representative compounds in nature. Depending on the molecular interactions with protein environment, these chromophores show a variety of photo‐absorption/emission energies. The present talk summarizes some recent theoretical studies (1) on the spectral tuning mechanism in photobiology using quantum chemical calculations with the symmetry‐adapted cluster configuration interaction (SAC‐CI) method. SAC‐CI is a coupled‐cluster method for describing the electron correlations in the excited states. The SAC‐CI method was introduced into the quantum mechanics (QM)/molecular mechanics (MM) framework for investigating excited states of chromophores and fluorophores in proteins. We studied spectral tuning mechanism of retinal proteins for human color vision (2) and emission color tuning mechanism of firefly luciferase (3) and fluorescent proteins (4). In these studies, transition energy was analyzed in terms of structural, environmental electrostatic, counter ion QM, and environmental QM effects. A common feature found in our study is (i) that the electronic transition involves an intra‐molecular charge‐transfer character and (ii) that the protein‐electrostatic potential (ESP) in the chromophore binding site is non‐uniform, rather polarized. Therefore, transition energy is controlled by the protein ESP through the positions of the amino acid residues. Analyzing the electrostatic interactions, we could also clarify amino acids' contributions to the spectral tuning. On the basis of the mechanisms, we proposed mutations for artificially controlling the color of proteins, which was computationally examined by computational simulations (3,4). References Hasegawa J, Fujimoto K, Nakatsuji H. Color tuning in photo‐functional proteins. ChemPhysChem, in press. Fujimoto K, Hasegawa J, Nakatsuji H. Color tuning mechanism in human red, green, and blue cone visual pigments: SAC‐CI theoretical study. Bull Chem Soc Jpn 2009;82:1140–8. Nakatani N, Hasegawa J, Nakatsuji H. Red Light in Chemiluminescence and Yellow‐green Light in Bioluminescence: Color‐tuning Mechanism of Firefly, Photinus pyralis, studied by the SAC‐CI method. J Am Chem Soc 2007;129:8756–65. Hasegawa J, Ise T, Fujimoto K, Kikuchi A, Fukumura E, Miyawaki A, Shiro Y. Excited States of Fluorescent Proteins, mKO and DsRed: Chromophore‐protein Electrostatic Interaction Behind the Color Variations. J Phys Chem B 2010;114:2971–9. Synthesis of highly chemiluminescent graphene oxide/metal nanoparticles nano‐composites and their analytical applications Yi He and Hua Cui* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, China E‐mail: hcui@ustc.edu.cn, heyi037@mail.ustc.edu.cn Graphene oxide/ noble metal nanoparticles nano‐composites exhibit unique properties such as high catalytic activity, good plasmonic property, effective bactericidal properties, and good biocompatible performance, which have been applied in sensors, biomedicine, catalysis and so on. Nevertheless, to the best of our knowledge, there is no any report about the graphene oxide/ noble metal nanoparticles nano‐composites with CL property to date. They are highly desirable due to their atomically flat surface, unique CL property, and excellent catalytic property, which are ideal candidates for nanoscale assembly. They may be used as advanced building blocks, CL labels, and platforms for various analytical devices with CL detection such as sensors, microchips and bioassays. Hence, it is of great interest to develop a new strategy for the synthesis of GO / noble metal nanoparticles with CL activity. In our present work, we reported a novel one‐step method at room temperature to prepare graphene oxide /metal nanoparticles (GO‐MNPs) nano‐composites with high chemiluminescence (CL) activity. The method is simple, fast, reliable, and linker free. The results of the characterization demonstrated that many MNPs were uniformly dispersed on the surface of GO nanosheets. And CL molecules were also decorated on the surface of nano‐composites. Thus nano‐composites exhibited good CL activity when reacting with oxidants such as H2O2. Furthermore, the formation mechanism of GO‐MNPs nano‐composites is also discussed. Finally, the effect of some biologically important small molecules such as glutathione, cysteine, homocysteine, tyrosine, and ascorbic acid on the CL reaction of obtained GO‐MNPs nano‐composites with H2O2 and its analytical application were explored. This work for the first time reported the GO/metal nanoparticles nano‐composites with CL activity. The nano‐composites may find future applications in the fields such as sensors, microchips and bioassays. References He Y, Liu DH, He XY, Cui* H. Chem. Commun. 2011;47:10692–4. Xu WP, Zhang LC, Li JP, et al. J. Mater. Chem. 2011;21:4593–7. Ren W, Fang Y, Wang E. ACS Nano 2011;5:6425–33. Using different kinds of fluorescence to determine the effect of herbicides, fungicides or pesticides on plants Angela Brüxa, Manfred Henneckea, Gadi Millerb and Stein Roaldsetc aBERTHOLD TECHNOLOGIES GmbH & Co. KG, Calmbacher Strasse 22, 75323 Bad Wildbad bMina and Everard Goodman Faculty of Life Sciences, Bar‐Ilan‐Universität, Ramat Gan, Tel Aviv, Israel cBERTHOLD TECHNOLOGIES U.S.A. LLC, 99 Midway Lane, Oak Ridge, TN 37830, USA The worldwide use of herbicides, fungicides or pesticides puts a costly burden on the farmers and leads to huge problems for the environment. Therefore it is of great importance to optimize the usage of these substances. The NightSHADE in vivo imaging system offers the possibility to monitor the influence and distribution of toxic substances such as herbicides on plants by measuring different kind of fluorescence. For example spray patterns can be improved and visualized by using fluorescent substances, whereas the plant stress status, cell death and chlorophyll content of the plant can be monitored by measuring emitted biophotons or determine delayed fluorescence. Here we show that all three kinds of fluorescence measurements can be easily combined in one application using the NightSHADE, allowing even kinetic measurements of the entire process. Stability and color shift of Photinus pyralis firefly luciferase upon introduction of sequential disulphide bridges Saman Hosseinkhani, Mahboobeh Nazari and Mehdi Imani Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Tel: (98)‐21‐82884407; Fax: (98)‐21‐82884484; E‐mail: (saman_h@modares.ac.ir) Firefly luciferase‐catalyzed reaction proceeds via the initial formation of an enzyme‐bound luciferyl adenylate intermediate. Multi‐color bioluminescence is developed using introduction of single/double disulphide bridges in firefly luciferase. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Single and double disulphide bridge is introduced into Photinus pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (A103C‐S121C, A296C‐A326C, C81‐A105C, L306C‐L309C, P451C‐V469C; C81‐A105C/ P451C‐V469C, and A296C‐A326C/ P451C‐V469C). By introduction of disulphide bridges using site‐directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red or kept in different extents. Multicolor bioluminescence is accompanied with displacement of a critical loop in red‐emitter luciferases without any shift in green emitters. Among mutants, A296C/A326C showed significantly increased thermostability, pH‐insensitivity and increased specific activity. Moreover, bioluminescence emission spectrum of A296C/A326C showed a resistance against physiologic temperature (37 °C), suggesting a useful reporter for several application, especially in the field of in vitro diagnostics and acidic environment. References Imani M, Hosseinkhani S, Ahmadian S, Nazari M. Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH sensitivity. Photochem Photobiol Sci. 2010;9(8):1167–77. Nazari M, Hosseinkhani S. Design of disulfide bridge as an alternative mechanism for color shift in firefly luciferase and development of secreted luciferase. Photochem Photobiol Sci. 2011;10(7):1203–15. The bioluminescence and fluorescence emission spectra of psychrofilic bacteria Photobacterium phosphoreum Kristina Alenina, Aleksey Loktushkin, Larisa Solov'eva and Anvar Ismailov Lomonosov Moscow State University, Faculty of Biology, Moscow, Russia E‐mail: anvaris@list.ru We have shown previously that psychrophilic bacteria Photobacterium phosphoreum, isolated from a gut of fish at a surface water (White Sea, 66°34′N, 33°08′E), produce at the low temperature in vivo highly intense bioluminescence with emission maximum about 478–480 nm with a shoulders at 500–510 and 530–540 nm. The emission spectrum can be attributed to the different accessory proteins that form a complex with luciferase involved in bioluminescence process. The aim of this study was the analysis of the spectral distribution of the bioluminescence in vivo and fluorescence of cell‐free extract of bacteria P. phosphoreum. Bacterial strain P. phosphoreum (KM MGU №331) from the Myoxocephalus scorpius was found in the coastal waters of the Kandalaksha's bay at the White Sea. Bioluminescence and fluorescence emission and excitation spectra (Fig. 1) were measured with the Jobin Yvon JY3C fluorescence spectrometer. Figure 1. Fluorescence emission (‐‐‐‐‐) and excitation (_____ ) spectra of P. phosphoreum cell‐free extract in 50 мM phosphat buffer + 2% NaCl, pH 7,5. With the excitation at 460nm, it was one emission peak at 540 nm. And with excitation at 380 nm, two peaks at about 450 nm and 510 nm with a shoulder near 530 nm, were observed. The fluorescent excitation spectra, with λem = 460, has a maximum at 350 nm, and a shoulders near 360 nm and 380 nm. With emission maximum at 530 nm, the excitation spectrum exhibits the dominant peak at 470 nm and second peak at 455 nm. It can be assumed, that psychrophilic photobacterium strain from the fish of White Sea (10–14 °C in summer), produces in vivo a complex spectrum of bioluminescence at 4–20 °C. Spectral distribution was made by using Origin 5 program (fig. 2). The basic spectrum with maximum about 480 nm can be formed as a superposition of three spectra of distinct color, include luciferase emitter (490) and accessory fluorescent proteins (460 nm and 530 nm) that participate in luminescence process. Figure 2. Bioluminescence emission spectra of the P. phosphoreum intact cells harvested from liquid culture and its spectral distribution in 50 мM phosphat buffer + 2% NaCl, pH 7,5. References Eckstein J, Cho K, Colepicolo P, Ghisla S, Hastings J, Wilson T. Proc. Natl. Acad. Sci. USA. 1990;87:1466–70. Karatani H, Wilson T, Hastings J. Photochemistry and photobiology 1992;55(2):293–9. Kuts VV, Ismailov AD. Microbiology‐(Russia) 2009;78(5):554–8. Karatani H, Matsumoto Sh, Miyata K, Yoshizawa S, Suhama Y, Hirayama S. Photochemistry and photobiology 2006;82:587–92. New evidence to “burglar‐alarm” function of bioluminescence in the ophiuroid species Ophiopsila aranea A Jones and J Mallefet(Marine Biology Laboratory, UCL, Belgium) Bioluminescence, the production of visible light by organisms, is widespread in the marine environment (1). Brittle stars are excellent models to study ecological functions of bioluminescence because of the remarkable abundance of luminous species in this class of echinoderms (66 luminous species known on 192 species tested (2)). Defensive functions of bioluminescence are commonly attributed to benthic luminous invertebrates (3). Among these functions, burglar alarm effect is defined as the use of light to attract a secondary predator during the attack of a primary predator (4). This function has been highlighted in the dinoflagellates Lingulodinum polyedrum (5), and suggested in the holothurian species Enypniastes eximia (6). We investigated this function in the brittle star Ophiopsila aranea, collected in Banyuls‐sur‐Mer (France). This species emits green intense flashes when disturbed, what makes it a good candidate for the burglar alarm function. Predators of two levels where chosen: the crab Carcinus maenas as primary predator, and the fish Diplodus vulgaris as secondary predator. Figure 1. Predation rates on the primary predator C. maenas during 12h long day or night interactions (n=6 for each combination) *P‐val < 0.05. Figure 2. Predation rates on the luminous brittle star O. aranea during 12h long day or night interaction (n=6 for each combination) *P‐val < 0.05. Predatory rate of the primary predator and of the brittle stars were recorded. Results concerning the primary predator support the burglar alarm hypothesis (Fig 1). Indeed, during the day, we observe that predation rate on the primary predator is higher when a brittle star is present, no matter if the brittle star is luminous or not. On the contrary, during the night, the predation on the primary predator is higher if O. aranea is present. It clearly indicates that light can be used by the fish to detect crabs when it attacks the luminous brittle star. We also recorded predation rates on O. aranea (Fig. 2). In this case, we do not observe any difference between combinations. The predation rate on the brittle star is lower during night than day, but does not decrease when predators of the two levels are present contrary to what is described in the theory (i.e. lower predation rate on the luminous brittle star due to a higher predation on the primary predator). These results give us support to the use of bioluminescence as burglar‐alarm signal for O. aranea, but we suggest that the benefits of this function are visible at the level of the population not for the single individual. Studies of the genetic structure of the Banyuls O. aranea population should in the future allow us to determine if a kin‐selected function of bioluminescence is conceivable for this species. This work was supported by the financial support ASSEMBLE grant agreement no. 227799. A. Jones is FNRS‐FRIA PhD student. J. Mallefet is a research associate of the FNRS. References Hastings JW. Cell Physiology Source Book, ACA press, San Diego, 1995;665–81. Mallefet J. Echinoderm bioluminescence. In Bioluminescence in Focus – A Collection of Illuminating Essays (Eds. Victor Benno Meyer‐Rochow), Research Signpost, India 2009;67–83. Herring PJ. Bioluminescent echinoderms: Unity of function in diversity of expression? Echinoderm Research 1995;9–17. Morin JG. Coastal bioluminescence: Patterns and Functions. Bul Mar Sci. 1983;33(4):787–817. Abrahams MV, TOWNSEND LD. Bioluminescence in dinoflagellates ‐ A test of the Burglar alarm Hypothesis. Ecology 1993;74:258–60. Rbison BH. Bioluminescence in the benthopelagic holothurian Enypniastes eximia. J. Mar. Bio. Ass. U.K. 1992;72:463–72. Development of novel telomerase assay by bioluminescent detection method K. Karasawa, Y. Sano and H. Arakawa School of Pharmacy, Showa University, Shinagawa‐ku, Tokyo 142‐8555, Japan Introduction Telomeres are specific structures found at the end of chromosomes in eukaryotes. In human chromosomes, telomeres consist of thousands of copies of 6 base repeats (TTAGGG) 1. Although human somatic cells induce cell‐death by reduction of telomeric repeats with cell division, cancer cells induce extension of telomeric repeats by telomerase. Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3' end of existing telomeres using its RNA component as a template. Therefore, telomerase participates in malignant transformation or immortalization of a cell, and attracts attention as anticancer drug screening and diagnostic tumor marker. Recently, telomeric repeat amplification protocol (TRAP) is used as universal method of telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplifying telomeric repeat by polymerase chain reaction (PCR); as a result, the TRAP method requires considerable time and skill for us. In this study, for rapid and high sensitive detection of telomerase activity, we developed novel telomerase assay using bioluminescent detection method; that is, pyrophosphates produced by telomerase reaction and PCR are converted to ATP by pyruvate phosphate dikinase (PPDK), and ATP is detected by firefly luciferin‐luciferase reaction2–3. Results and discussion The reaction scheme is shown in Fig. 1. In this study, the detection limit of pyrophosphate was 1.0 × 10−15 mol/assay. For optimal bioluminescent assay of telomerase activity, we designed the specific primers to the telomeric repeat and selected the efficient Taq polymerase for PCR. Sequences of the sense and antisense primers for PCR amplification of telomerase reaction product were 5′‐AATCCGTCGAGCAGAGTT‐3' and 5'‐ CTAACCCTAACCCTAACC‐3′, respectively. In study of Taq polymerase, efficient PCR amplification could be obtained by use of TITANIUM Taq DNA polymerase. After the telomerase reaction and subsequent PCR, the released pyrophosphate was detected by the proposed bioluminescent assay. As a result, positive cell (500 cells) and inactive cell (prepared by heating at 85 °C for 10 min) could be clearly identified. Then, time course of bioluminescent intensity was examined. As a result, the maximum bioluminescence intensity was maintained for about two minutes. The detection limit of cells with telomerase was examined with 500, 250, 100, 50, 10, 5, 1 cell. As a result, 1 cell/assay was detectable by telomerase reaction for 30 minutes and PCR consisting of 33 cycles. PCR cycle number also was examined, 25 cycles was detectable. Presently, we are examining simpler and rapid bioluminescent detecting method, and we are developing its application in clinical chemistry of cancer and in basic research such as regenerative medicine. Figure 1. Reaction scheme of the telomerase assay. References Blackburn EH. Structure and function of telomeres. Nature 1991;350:569–73. Arakawa H, Karasawa K, Igarashi T, Suzuki S, Goto N, Maeda M. Anal Biochem. 2004;333(2):296–302. Arakawa H, Karasawa K, Munakata E, Obinata R, Maeda M, Suzuki S, Kamahori M, Kambara H. Anal Biochem 2008;379:86–90. Enantioselective thioesterification activity in bioluminescene enzyme, firefly luciferase Dai‐ichiro Kato, Yoshihiro Hiraishi, Keisuke Yokoyama, Kazuki Niwa, Yoshihiro Ohmiya, Masahiro Takeo and Seiji Negoro University of Hyogo, 2167 Shosha, Himeji, Hyogo, 671‐2280 Japan E‐mail: kato@eng.u‐hyogo.ac.jp Firefly luciferase catalyzes the oxidation of D‐luciferin with molecular oxygen in the presence of ATP and Mg2+, producing light. Firefly luciferase, however, exhibits bimodal action depending on the substrate. In the presence of ATP, Mg2+, and coenzyme A (CoASH), this enzyme converts the non‐luminescent substrates L‐luciferin and dehydroluciferin into the corresponding thioesters. This catalytic activity allows firefly luciferase to release the inhibitive non‐luminous substrate, resulting in an overall enhancement of the bioluminescence reaction. Interestingly, this activity is also displayed toward quite different compounds such as long chain fatty acids. This fact stirred us up to investigate the application of firefly luciferase to synthetic substrates. We noticed the resemblance between firefly luciferases and long‐chain acyl‐CoA synthetases (LACS). LACS can catalyze the enantioselective thioesterification of 2‐arylpropanoic acids(1). Based on the sequence and the reaction mechanisms similarities between firefly luciferase and LACS(2), we predicted that the thioesterification activity of firefly luciferases would display enantioselectivity. Our objective has been achieved partially, we could have confirmed some firefly luciferases such as from Luciola lateralis (LUC‐H) and Pylocoeria miyako (PmL) catalyze the (R)‐enantioselective thioesterification of a series of 2‐arylpropanoic acids (Fig. 1) (3,4). On the other hand, other luciferases such as from Luciola mingrelica (LmL) and Hotaria parvura (HpL) could not recognize these compounds(5). Because all these luciferases exhibit the thioesterification activity toward fatty acids, these are thought to come from the differences of the substrate recognition system in each luciferase. Figure 1. Firefly luciferase catalyzed (R)‐enantioselective thioesterification of 2‐arylpropanoic acid (ketoprofen). In this study, we will report some important residues to define the substrate specificity in this enzyme. Using PmL and HpL as model enzyme, we could convert the reciprocal recognition ability toward 2‐arylprpanoic acid. In addition, the differences around the catalytic site were estimated by molecular dynamics calculation method. These data provide us with the useful information of substrate recognition network when firefly luciferase acts as the thioesterification enzyme. References Knihinicki RD, Williams KM, Day RO. Chiral Inversion of 2‐Arylpropionic Acid Nonsterpodal Anti‐Inflammatory Drugs‐1 In Vitro studies of Ibuprofen and Flurbiprofen, Biochem. Pharmacol. 1989;38:4389–95. Suzuki H, Kawarabayashi Y, Kondo J, Abe T, Nishikawa K, Kimura S, Hashimoto T, Yamamoto T. Structure and Regulation of Rat Long‐chain Acyl‐CoA Synthetase, J. Biol. Chem. 1990;256:8681–5. Kato D, Teruya K, Yoshida H, Takeo M, Negoro S, Ohta H. New application of firefly luciferase ‐ it can catalyze the enantioselective thioester formation of 2‐arylpropanoic acid, FEBS J. 2007, 274, 3877–85. Kato D, Tatsumi T, Bansho A, Teruya K, Yoshida H, Takeo M, Negoro S. Enantiodifferentiation of ketoprofen by Japanese firefly luciferase from Luciola lateralis, J. Mol. Catal. B: Enzmatic 2011;69:140–6. Kato D, Yokoyama K, Hiraishi Y, Takeo M, Negoro S. Comparison of Acyl‐CoA Synthetic Activities and Enantioselectivity toward 2‐Arylpropanoic Acids in Firefly Luciferases, Biosci. Biotech. Biochem. 2011;75:1758–62. Chemiluminescence of 1,2,4‐trioxolanes and 1,2,4,5‐tetroxanes: fundamentals and possible biomedical applications Dmitri V. Kazakova, Farit E. Safarova, Timur A. Nazirova, Oxana B. Kazakovaa, Alexandr O. Terent'evb, Dmitri A. Borisovb and Waldemar Adamc aInstitute of Organic Chemistry, Ufa Scientific Center of the RAS, 71 Pr. Oktyabrya, 450054 Ufa, Russia bZelinsky Institute of Organic Chemistry of the RAS, 47 Leninskiy prospekt,119991 Moscow, Russia cDepartment of Chemistry, Facundo Bueso 110, University of Puerto Rico, Rio Piedras, Puerto Rico 00931, USA and Institut für Organische Chemie der Universität Würzburg, Am Hubland The 1,2,4‐trioxolane and 1,2,4,5‐tetroxane pharmacophores are currently considered as the next generation of synthetic antimalarial drugs(1,2). We report here on the light emission of these cyclic peroxides in their reactions with Fe(II). This constitutes a bio‐medically important discovery since the anti‐malarial activity of peroxides is presumed to be mediated by Fe(II)‐induced cleavage of peroxide bond. The chemiluminescence has been induced by FeSO4 and/or FeCl3/L‐cysteine/rhodamine G system in aqueous (50%) acetonitrile. The light emission in the visible spectral region has been recorded for the triterpenoid‐based 1,2,4‐trioxolanes 1 and 2, the purely synthetic ozonide OZ03, the bicyclic 1,2,4,5‐tetroxanes 3 and 4, the tetroxane derived from deoxycholic acid 5, the diperoxide of trifluoroacetone 6, as well as the natural artemisinin(3,4): Our herein discovered chemiluminescence provides promising perspectives for the study of pharmacologically active peroxides in biomedical applications. References Vennerstrom JL, Arbe‐Barnes S, Brun R, Charman SA, Chiu FCK, Chollet J, Dong Y, Dorn A, Hunziker D, Matile H, McIntosh K, Padmanilayam M, Tomas JS, Scheurer C, Scorneaux B, Tang Y, Urwyler H, Wittlin S, Charman WN. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 2004;430:900. Muraleedharan KM, Avery MA. Progress in the development of peroxide‐based anti‐parasitic agents. Drug Discovery Today 2009;14:793. Kazakov DV, Timerbaev AR, Safarov FE, Nazirov TI, Kazakova OB, Ishmuratov GY, Terent'ev AO, Borisov DA, Tolstikov AG, Tolstikov GA, Adam W. Chemiluminescence from the biomimetic reaction of 1,2,4‐trioxolanes and 1,2,4,5‐tetroxanes with ferrous ions. RSC Adv., 2012, DOI: 10.1039/C1RA00784J Kazakova OB, Kazakov DV, Yamansarov EYu, Medvedeva NI, Tolstikov GA, Suponitsky KYu, Arkhipov DE. Synthesis of triterpenoid‐based 1,2,4‐trioxolanes and 1,2,4‐dioxazolidines by ozonolysis of allobetulin derivatives. Tetrahedron Lett. 2011;52:976. Enhancing chemiluminescence reaction of luminol from specific species of Lumbricus rubellus earthworm MJ Chaichi*, A Khodabandeh, R Akhoondi, A Esmaeili and M Parvar Faculty of Chemistry, University of Mazandaran, Babolsar, Iran E‐mail: jchaichi@yahoo.com The coelomic cavity of earthworms may be inhabited by various sorts of soil‐derived parasites, such as bacteria, gregarines and fungi, which are kept in check by the combined activities of the earthworm coelomocytes and humoral factors (1). Analysis by phase‐contrast fluorescent microscopy and flow cytometry demonstrated that eleocytes of some earthworm species exhibit a strong auto‐fluorescence. The main fluorophore responsible for eleocyte fluorescence was indicated that riboflavin but not FMN (flavin mononucleotide) or FAD (flavin‐adenine dinucleotide) (2). Earthworms have many complex relationships with soil. The effect of Pb and Zn on coelomocyte riboflavin content in the epigeic earthworm Dendrodrilus rubidus was measured by flow cytometry and spectrofluorimetry. Excessive essential or nonessential metal exposures interfere with earthworm performance at all levels of biological organisation, from demographic parameters and cellular integrity, to metabolome and transcriptome profiles (3). Chemiluminescence (CL) has attracted considerable attention as a versatile and highly sensitive detection tool within diverse fields such as biology, biotechnology and analytical technology. Luminol is oxidized by strong oxidants in the presence of a catalyst such as peroxidase to produce chemiluminescence, leading to its use in a variety of analytical methods (4). The main aim of the present study was to measure the riboflavin content in autofluorescent eleocytes of the small ‘gilttailed’ earthworm species Lumbricus rubellus sampled. The extracted species directly were added to CL reaction of luminol and hydrogen peroxide in presence of hemoglobin. It was convincingly showed the enhancement of chemiluminescence intensity of luminol chemiluminescence system (luminol–H2O2–hemoglobin) in the presence of specific species of earthworm L. rubellus. Results also introduced a reliable method for determination of riboflavin content in eleocytes by using luminol‐ luminescence system. References Olchawa E, Bzowska M, Stiirzenbaum SR, Morgan AJ, Plytycz B. (2006) Heavy metals affect the coelomocyte‐bacteria balance in earthworms: Environmental interactions between abiotic and biotic stressors. Environ. Pollut 2006:142:373–81. Plytycz B, Homa J, Koziol B, Rozanowska M, Morgan AJ. Riboflavin content in autofluorescent earthworm coelomocytes is species‐specific. Folia Histochemica et Cytobiologica 2006:44(4):275–80. Plytycz B, Lis‐Molenda U, Cygal M, Kielbasa E, Grebosz A, Duchnowski M, Andre J, Morgan AJ. Riboflavin content of coelomocytes in earthworm (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution. Environmental Pollution 2009:157:42–3050. Mestre YF, Zamora LL, Calatayud JM. Flow‐chemiluminescence: a growing modality of pharmaceutical analysis. Luminescence 2001:16:213–35. Interactions of halogenated compounds with bioluminescent enzymes TN Kirillovaa, MA Gerasimovab, EV Nemtsevaa,b and NS Kudryashevaa,b aInstitute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia bSiberian Federal University, Krasnoyarsk, 660041, Russia Effect of heavy halogen atom on bioluminescent (BL) reactions, i.e. inhibition of BL intensity by halogenated compounds ‐ bromides and iodines, was demonstrated earlier (1) in reactions of luminous bacteria, fireflies and coelenterates. Quenching efficiencies of the halogenated compounds in BL and photoluminescence of model emitters were compared. Based on the results of this comparison, a conclusion was made that contribution to BL quenching of enzyme‐halogen interactions (biochemical mechanism) is higher, than that of Br and I effects on electron‐excited states of BL emitters (physical mechanism). Purpose of the current study was to reveal “the effect of heavy halogen atom” in binding of halogenated compounds with enzymes of luminous organisms ‐ fireflies Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima. Homologous xanthene dyes (fluorescein, eosin, and erythrosin) with halogen substituents of different atomic weight were applied as model fluorescent markers. The enzyme‐dye interactions were analyzed using fluorescence characteristics of the dyes in steady‐state and time‐resolved experiments. Dependences of (1) fluorescence anisotropy of enzyme‐bound dyes, (2) average fluorescence lifetime, and (2) number of exponential components in fluorescence decay, on atomic weight of halogen substituents were demonstrated. Fig. 1 presents fluorescence anisotropy of xanthene solutions at different concentrations of firefly luciferase, taken as an example (2). It is seen that in a row of dyes fluorescein‐eosin‐erythrosin, the r‐values increase at all luciferase concentrations, simultaneously with atomic weight of halogen substituents in the dye molecules. Figure 1. Fluorescence anisotropy r of solutions of fluorescein (1), eosin (2), and erythrosin (3) (C = 1 µmol L‐1) at different concentrations C of firefly luciferase. The effects were attributed to the “dark” process, namely, to the interaction of halogenated compounds with enzymes, followed by excited BL emitter formation; hydrophobic interactions were assumed to be responsible for this effect. Firefly luciferase was found to be the most effective enzyme in dye–enzyme binding interactions. Acknowledgements The work was supported by Grants Ministry of Education RF N2.2.2.2/5309, ‘Leading Scientific School’ N 1211.2008.4; Program ‘Molecular&Cellular Biology’ of RAS. References Kirillova TN, Kudryasheva NS. Effect of heavy atoms in bioluminescent reactions. Anal. Bioanal. Chem. 2007;387:2009–16. Kirillova TN, Gerasimova MA, Nemtseva EV, Kudryasheva NS. Effect of halogenated fluorescent compounds on bioluminescent reactions. Anal. Bioanal. Chem. 2011;400(2):343–51. General toxicity of heavy metal solutions in the presence of humic substances. Bioluminescent monitoring SL Kislana, AS Tarasovab and NS Kudryashevaa,b aSiberian Federal University, Krasnoyarsk, Russia bInstitute of Biophysics SB RAS, Krasnoyarsk, Russia Salts of heavy metals are among the most common toxic pollutants. Humic substances (HS), being products of oxidative degradation and polymerization of organic matter in soils, can serve as natural detoxifying agents in solutions of metal salts in nature (1). Figure 1. Bioluminescence intensity (IHS) vs. HS concentration (CHS) in solution of CoCl2. 5∙10‐3 M. Enzymatic assay. Toxicity of a series of salts of metals (chromium, cobalt, copper, europium, lead) was studied in the presence and absence of HS in model water solutions. Two bioluminescent assay systems were applied to monitor toxicity of the solutions: luminous bacteria Photobacterium phosphoreum and bioluminescent system of coupled enzymatic reactions catalyzed by bacterial luciferase and oxidoreductase. Dependences of bioluminescent intensity on HS concentration in the salts solutions were studied. Example of this dependence in CoCl2 solutions is shown in Fig. 1. Low HS concentrations were found to increase bioluminescent intensity, thus producing the “detoxifying effect”(IHS > 1. Fig. 1). Detoxification coefficients, D, were calculated to characterize changes in toxicity of solutions under HS action: D=Ihs/I, where IHS and I are maximal bioluminescent intensities in salt solutions in the presence and absence of HS, respectively. High HS concentrations inhibited bioluminescence, revealing increase of toxicity. As is seen from Fig. 1, the threshold concentration of HS was 0.002 g/L in CoCl2 solution. It was shown, that D values obtained by bacterial assay were higher than those of the enzymatic assay. This is an evidence of bacterial adaptation to toxic effect of metal salt in HS solutions and active role of bioassay systems in toxicity definition. It was demonstrated that increase of time of metal exposure to HS resulted in toxicity decrease (increase of D values). This effect is probably concerned with low rates of HS‐metal complex formation in water solutions. References Bollag J‐M, Mayers K. Detoxification of aquatic and terrestrial sites through binding of pollutants to humic substances. Sci. Total Environ. 1992;117/118:357–66. Tarasova AS, Stom DI, Kudryasheva NS. Effect of humic substances on toxicity of oxidizer solutions. Environ. Toxic. Chem. 2011;30:1013–7. Combined effect of mutations stabilizing green and red emitters on bioluminescence of firefly luciferase MI Koksharov and NN Ugarova Dept. of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia E‐mail: mkoksharov@gmail.com Most wild‐type firefly luciferases demonstrate highly pH‐sensitive bioluminescence spectra, which undergo a large shift from green to red light when lowering pH from 7.8 to 6.0. Similar shifts are also observed at elevated temperatures. This red shift is attributed to the switching between two different molecular forms of the product (green and red emitters) in the bimodal spectrum of luciferase. However, the precise structural mechanism that determines the bioluminescence color of firefly luciferase is still unknown. A large number of mutants were discovered in the last 20 years that change the profile of the bioluminescence spectra. Some of them shift the maximum of the individual spectral components but most mutations affect the ratio between the two forms of the emitter. Several mutants of Luciola mingrelica luciferase affecting the color and pH‐sensitivity of its bioluminescence spectra were earlier characterized in our laboratory. The mutation H433Y shifted the color of bioluminescence to red by greatly increasing the contribution of the red emitter to the bimodal spectra (1). On the contrary, the mutants Y35N and Y35H greatly stabilized the green emission preventing the essential increase of the red emission at low pH or high temperatures (2) (Fig. 1). The mutant A217T/S222T was also obtained during the random mutagenesis experiments described the latter work. It demonstrated a large shoulder in the red region. Thus, it was interesting to what extent the mutation Y35N, which stabilizes the green emitter, can compensate the strong red‐shifting effect of the mutant H433Y and the weaker effect of the mutant A217T/S222T. Figure 1. Bioluminescence spectra of WT (1) luciferase and mutants Y35N (2), H433Y (3), Y35N/H433Y (4), ‘7MT1’ (5), Y35N/‘7MT1’ (6) at pH 7.8 (25 °C), pH 6.0 (25 °C) and at 10 °C (pH 7.8). Figure 2. In vivo bioluminescence of E. coli colonies producing WT luciferase and the mutants Y35N, H433Y and Y35N/H433Y. At the first part of this work the mutants Y35N/H433Y and Y35N/A217T/S222T were constructed and characterized. The bioluminescence spectra and pH‐sensitivity of the latter mutant were similar to that of the mutant Y35N. In the case of the former, the mutations Y35N and H433Y compensated each other: the double mutant Y35N/H433Y demonstrated yellow‐green bioluminescence in vitro (Fig. 1) and yellow‐orange bioluminescence in vivo in E. coli colonies (Fig. 2) like the wild‐type (WT) luciferase. We decided to use this compensating ability of the mutant Y35N to find new strong red‐shifting mutants in the C‐terminal domain of luciferase (442–548 aa residues). This area was subjected to random mutagenesis and screening, while the parent enzyme contained the mutation Y35N. The red‐shifting mutant designated ‘7MT1’ was identified. When applied to WT, this mutation significantly increased the contribution of the red emitter, thus changing the maximum from 566 to 604 nm, which is similar to the effect of H433Y. However, this substitution also caused the 12 nm red shift of the green emitter (from 566 to 578 nm), which is evident from the bioluminescence spectra at low temperatures and the spectra of the double mutant Y35N/‘7MT1’ (Fig. 1). Structural reasons for the effect of this mutation are discussed. The mutants that strongly stabilize the green or red emitter of firefly luciferase can be an efficient tool to identify new important positions affecting bioluminescence color by sequential scanning the enzyme structure through random mutagenesis. This can help to elucidate the mechanism of the color determination in beetle luciferases and develop new enzymes for multi‐color luciferase applications. References Ugarova N, Maloshenok L, Uporov I and Koksharov M. Bioluminescence spectra of native and mutant firefly luciferases as a function of pH. Biochemistry (Moscow). 2005;70:1262–7. Koksharov MI, Ugarova NN. Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes whose bioluminescence spectra show low pH‐sensitivity. Biochemistry (Moscow). 2008;73:862‐9. Effect of the substitutions G216N/A217L and S398M on thermal stability, activity and bioluminescence color of L. mingrelica firefly luciferase MI Koksharov and NN Ugarova Dept. of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia E‐mail: mkoksharov@gmail.com Insufficient thermostability of wild‐type (WT) firefly luciferases often limits their application. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases, for example, Luciola lateralis (Lll), Luciola cruciata (Lcl) and Photinus pyralis (Ppl) luciferases (1). However, for Hotaria parvula firefly luciferase (Hpl), which shares 98% sequence identity with Luciola mingrelica luciferase (Lml), the A217L mutation is known to dramatically decrease catalytic activity more than 1000‐fold (1). We have analyzed the environment of A217 in the 3D‐structure of Lml and compared it with that in Hpl, Lll, Lcl, Ppl in order to propose possible additional mutations that would retain the high thermal stability of the mutant A217L while preserving the high level of activity. The 7Å environment of A217 is identical in Lml and Hpl, thus it is safe to assume that in both these highly homologous enzymes the single substitution A217L would lead to the loss of activity. The neighboring residue G216 and the more remote S398 appeared to be the key positions that distinguish the environment of A217 in a small subgroup of luciferases including Lml and Hpl from that of Lll, Lcl, Ppl and most others. In other beetle luciferases the position 216 is occupied with a residue having a side group (in contrast to G216 in Lml) and the position 398 is generally occupied with methionine. We decided to eliminate these differences to make the A217 environment similar to that of Lcl and thus possibly prevent the loss of activity in the case of the substitution A217L in Lml. The double mutant G216N/A217L had a half‐life of 160 and 80 min at 42 °C and 45 °C, respectively, which is 18‐ and 28‐fold increase in stability over WT Lml. However, it retained only 10% of activity. The loss in activity was accompanied by a large red shift of the bioluminescence emission maximum from 566 to 611 nm compared with the wild type enzyme. This shift was caused by the significant increase of the contribution of the “red emitter” in the bimodal spectrum of firefly luciferase (Fig. 1). Interestingly, the red shift of this mutant and its bioluminescence spectra were similar to that of the mutant H433Y studied previously (2), which is located 23 Å away from the position 217. Since the change G216N was insufficient to obtain a fully active luciferase, the additional substitution S398M was introduced. The mutant S398M alone showed catalytic properties and stability similar to that of WT. Its bioluminescence spectra were slightly less pH‐ and temperature sensitive. The triple mutant G216N/A217L/S398M possessed the high thermal stability of the double mutant as well as high activity and yellow‐green bioluminescence of the wild‐type enzyme (3). Thus, the substitution S398M was able to effectively restore the activity and color of the mutant G216N/A217L (Fig. 1, Fig. 2). Figure 1. Bioluminescence spectra of WT (1) luciferase and the mutants S398M (2), G216N/A217L (3), G216N/A217L/S398M (4), H433Y (5) at pH 7.8 and pH 6.0 (25 °C). Figure 2. In vivo bioluminescence of E. coli colonies producing WT (1) luciferase and the mutants S398M (2), G216N/A217L (3), G216N/A217L/S398M (4). In conclusion it can be stated that rational protein design of a residue microenvironment can be an effective strategy when a single mutation does not lead to the desirable effect reported for the similar substitution in a homologous enzyme. References Kitayama A, Yoshizaki H, Ohmiya Y, Ueda H, Nagamune T. Creation of a thermostable firefly luciferase with pH‐insensitive luminescent color. Photochem. Photobiol. 2003;77:333–8. Ugarova N, Maloshenok L, Uporov I, Koksharov M. Bioluminescence spectra of native and mutant firefly luciferases as a function of pH. Biochemistry (Moscow). 2005;70:1262–7. Koksharov MI, Ugarova NN. Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelica firefly luciferase. Photochem. Photobiol. Sci. 2011;10:931–8. Simultaneous determination of SNP genotypes by photoprotein obelin and R. muelleri luciferase VV Krasitskaya, LP Burakova and LA Frank Photobiology Laboratory, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia Siberian Federal University, Krasnoyarsk 660041, Russia Primer extension reaction (PEXT) is the most widely used approach of single nucleotide polymorphisms (SNP) genotyping. We propose a dual‐analyte bioluminometric method for simultaneous detection of normal and mutant allele in a high sample‐throughput format. The recombinant Ca2+‐regulated photoprotein obelin and coelenterazine‐dependent luciferase Renilla muelleri were used as reporters. PCR‐amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin‐dUTP and biotin‐dUTP. Both primers contain a d(A)27 segment at the 5′‐end but differ in the final nucleotide at the 3'‐end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin‐ or biotin‐labeled product. The products of PEXT reactions are mixed, denatured and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of obelin‐antibody to digoxigenin conjugate and streptavidin‐luciferase R. muelleri conjugate. The flash‐type bioluminescent reaction of obelin is triggered by addition of Ca2+, bioluminescence of luciferase than measured by adding Ca2+‐triggered coelenterazine‐binding protein Renilla (Figure 1). The method was evaluated by analysis of single nucleotide polymorphism known as factor V Leiden 1691 G→A (R506Q). Clinical DNA samples were tested by the proposed method. The results agreed entirely with the RT‐PCR data. Figure 1. The principle of PEXT reaction and microtiter well‐based detection of PEXT products. B – biotin, D – digoxigenin, Obe – obelin, Luc – luciferase R. muelleri, St – streptavidin, CBP – Ca2+‐triggered coelenterazine‐binding protein Renilla, CE – coelenterazine, BSA – bovine serum albumin (BSA‐T30 conjugate), Y – antibodies to digoxigenin, ● – Ca2+. This work was supported by grant No. 76 of the Russian Academy of Sciences, Siberian Branch and by the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). Bioluminescent enzymatic biosensors: from idea to laboratory Valentina Kratasyuka,b and Elena Esimbekovab,a aSiberian Federal University, Krasnoyarsk, 660041, E‐mail: ValKrat@mail.ru bInstitute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia Current methods cannot solve the pressing problem of how to detect, identify and measure the contents of the numerous chemical compounds that differ in their physico‐chemical and toxic characteristics. The problem is important for environmental monitoring, food product monitoring and medical diagnostics. We proposed Bioluminescent Enzyme System Technology BESTTM (1,2), where the bacterial coupled enzyme system: NADH‐FMN oxidoreductase‐ luciferase substitutes for living organisms. In the presence of toxic agents, enzymes from luminous bacteria more closely reflect the toxicity of living organisms than does the use of chemical analysis. BESTTM was introduced to facilitate and accelerate the development of cost‐competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications. However, their wide‐spread use could be hindered by several disadvantages, including the instability of enzyme systems during use, limited shelf life, the need to control ambient conditions (i.e. pH and temperature), interference by substances in the sample and manufacturing cost. To solve these problems, a new method was developed to design polyenzymatic systems that incorporate multiple substrates with the oxidoreductase – luciferase enzymes isolated from a proprietary collection of luminous bacteria strains. A patented stabilization and immobilization process preserves up to 50% of the enzymatic activity and produces the homogeneous multi‐component reagent “Enzymolum”, which contains the bacterial luciferase, NADH‐FMN oxidoreductase and their substrates (1,2), co‐immobilized in starch and gelatin gel. The reagent is currently produced in tablet form and can be used only in the cuvette variant of a bioluminometer. The other forms, e.g. on the plane table, strips and others were also obtained for bioluminescent analysis. “Enzymolum” can be integrated as a biological module into the portable biodetector‐biosensor of original construction. “Enzymolum” is the central part of Portable Laboratory for Toxicity Detection (PLTT), which consists of a biological module, a biodetector module, a sampling module, a sample preparation module, and a reagent module. PLTT immediately signals chemical‐biological hazards and allows us a) to detect a wide range of toxic substances – more than 25,000 compounds; b) to perform express‐screening for toxicity in emergency situations in field and laboratory condition; c) to develop systems for analyzing individual compounds; d) to develop systems to evaluate the degree of whole toxicity; e) to keep the high sensitivity of reagents for many years; f) to perform biotesting at high concentrations of organic substances in water and g) to develop a portable biosensor for personal use. Prototype biosensors developed with this technology offer cost advantages, versatility, high sensitivity (up to 10−14 moles of analyte), rapid response time (less than 3 minutes), extensive shelf life (up to 5 years without loss of activity), and flexible storage conditions (up to +25 °C). The enzyme biotesting approach was used as a platform technology to certify “Method to measure the intensity of bioluminescence with the help of the “Enzymolum” reagent to detect the toxicity of drinking, natural, waste and treated waste water” (1). The laboratory will be the principle example of a whole family of new, portable, professional laboratories for local services of ecological monitoring, ecological laboratories in industrial corporations, state ecological departments, food quality laboratories, military departments and other monitoring, teaching, security and research organizations. This work was supported by the Federal Agency of Science and Innovations (contract No 02.740.11.0766), the Russian Academy of Sciences (Program “Molecular and cellular biology”, grant No 6.2), President of RF (grant Leading scientific school No 64987.2010.4), the Program of the Government of the Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). References Kratasyuk VA, Esimbekova EN. Patent RF No. 2413771 Express method for biotesting of natural, manufactoring waters and water solutions. Published on March 10, 2011. Kratasyuk VA, Esimbekova EN. Patent RF No. 2413772. Bioluminescent biomodul for analyses of various media toxicity and method of its preparation. Published on March 10, 2011. Quantum chemistry behind bioimaging: Insights from ab initio studies of fluorescent proteins and their chromophores Anna I. Krylov Dept. of Chemistry, University of Southern California, Los Angeles, CA 90089‐0482 The unique properties of green fluorescent protein (GFP) have been harnessed in a variety of bioimaging techniques, revolutionizing many areas of the life sciences. Molecular‐level understanding of the underlying photophysics provides an advantage in the design of new fluorescent proteins (FPs) with improved properties; however, because of its complexity, many aspects of the GFP photocycle remain unknown. This lecture will discuss computational studies of FPs and their chromophores that provide qualitative insights into mechanistic details of their photocycle and the structural basis for their optical properties. An interesting feature of several anionic FP chromophores in the gas phase is their low electron detachment energy. For example, the bright excited ππ* state of the model GFP chromophore (2.6 eV) lies above the electron detachment continuum (2.5 eV). Thus, the excited state is metastable with respect to electron detachment. This autoionizing character needs to be taken into account in interpreting gas‐phase measurements and is very difficult to describe computationally. Solvation (and even microsolvation by a single water molecule) stabilizes the anionic states enough such that the resonance excited state becomes bound. However, even in stabilizing environments (such as protein or solution), the anionic chromophores have relatively low oxidation potentials and can act as light‐induced electron donors. Protein appears to affect excitation energies very little (< 0.1 eV), but alters ionization or electron detachment energies by several electron volts. Solvents (especially polar ones) have a pronounced effect on the chromophore's electronic states; for example, the absorption wavelength changes considerably, the ground‐state barrier for cis–trans isomerization is reduced, and fluorescence quantum yield drops dramatically. Calculations reveal that these effects can be explained in terms of electrostatic interactions and polarization, as well as pecific interactions such as hydrogen bonding. The results of sophisticated first‐principle calculations can be interpreted in terms of simpler, qualitative molecular orbital models to explain general trends. In particular, an essential feature of the anionic GFP chromophore is an almost perfect resonance (mesomeric) interaction between two Lewis structures, giving rise to charge delocalization, bond‐order scrambling, and, most importantly, allylic frontier molecular orbitals spanning the methine bridge. We demonstrate that a three‐center Hückel‐like model provides a useful framework for understanding properties of FPs. It can explain changes in absorption wavelength upon protonation or other structural modifications of the chromophore, the magnitude of transition dipole moment, barriers to isomerization, and even non‐Condon effects in one‐ and two‐photon absorption. Reference Bravaya KB, Grigorenko BL, Nemukhin AV, Krylov AI. Quantum chemistry behind bioimaging: Insights from ab initio studies of fluorescent proteins and their chromophores, Acc. Chem. Res. in press (2011). Using of bioluminecent assay to monitor radioactive toxicity NS Kudryashevaa,b, MA Alexandrovab and TV Rohkob aInstitute of Biophysics SB RAS, Akademgorodok 50, 660036. Krasnoyarsk, Russia bSiberian Federal University, Svobodniy 79, 660041, Krasnoyarsk, Russia The bioluminescent (BL) bioassays are traditionally applied for monitoring of chemical toxicity. Main testing physiological parameter of the bioassays is BL intensity. Not long ago we used them for the first time to monitor radiation toxicity in solutions of alpha‐ (1.2) and beta‐ (3) radionuclides. The luminous bacteria serve as convenient models for studing effects of ionizing radiation on living organisms. The purpose of the work was to study chronic effects of radionuclides on glowing of luminous bacteria Photobacterium Phosphoreum. Effects of model solutions of alpha‐emitting nuclide Am‐241 and beta‐emitting nuclide tritium were studied. The bacteria were grown in nutrient media with addition of Am‐241 (up to 7 kBq/L), H3‐labled aminoacid valine, or tritiated water (up to 100 MBq/L). The Am‐241 inhibited bacterial growth at all activities of the nutrient media. The tritium increased bacterial growth at activity <30 MBq/L, and inhibited it at >30 MBq/L. Bacteria were sampled at exponential and stationary stages of growth; BL time‐course of the samples was studied and compared with that of a control (nonradioactive) sample. Three stages were found in BL kinetics of the radioactive samples of Am‐241 and tritium: (1) absence of the effect, (2) BL activation, and (3) BL inhibition. The BL activation reached 1000–2000%; it was attributed to hormesis phenomenon. All three BL kinetics stages were found in solutions of both Am‐241 and tritium, i.e. the response of the cells was unified. The stages of BL time‐course correspond to general regularity in responses of all organisms to stress‐factors: (1) identification of a stress‐ factor, (2) adaptive response/syndrome, (3) suppression of a physiological function. BL time‐course in the presence of Am‐241 and tritium was studied in BL enzymatic reactions. It was compared to that of bacterial BL. The results show that the resistance of the BL function to radionuclides increases from enzymes to cells, i.e. with increase of complexity of the systems. Accumulation of Am‐241 and tritium in bacterial cells and DNA was determined. Role of peroxides (as secondary products of ionizing radiation in water) in the effects radionuclides on luminous bacteria and their enzymatic reactions were studied. Peroxides were found in to be effective in Am‐241 solutions and were not – in tritium solutions (2). Acknowledgements The work was supported by Grants from RFBR N10‐05‐01059‐a Ministry of Education RF N2.2.2.2/5309, ‘Leading Scientific School’ N 1211.2008.4; Program ‘Molecular&Cellular Biology’ of RAS. References Rozhko TV, Kudryasheva NS, et al. Effect of low‐level α‐radiation on bioluminescent assay systems of various complexity. Photochem. Photobiol. Sci. 2007;6:67–70. Alexandrova M, Rozhko T, et al. Effect of americium‐241 on luminous bacteria. Role of peroxides. Journal of Environmental Radioactivity 2011;102,407–11. Alexandrova MA, Rozhko TV, et al. Effect of tritium on growth and bioluminescence of bacteria P.Phosphoreum. Radiat. Biol. Radioekol. 2010;6:613–8. Dual‐analyte single‐well bioluminescence immunoassay based on obelin color mutants AN Kudryavtseva,b, VV Krasitskayaa,b and LA Franka,b aPhotobiology Laboratory, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia bSiberian Federal University, Krasnoyarsk 660041, Russia Obelin mutants W92F‐H22E and Y138F are characterized by rather different bioluminescence: 1) the emission spectra maxima are separated by 103 nm, spectral overlap is small; 2) the decay rates of their signals differ as much as 10‐fold (1). These spectral and kinetic differences make possible the effective signals' separation using band‐pass optical filters and plate luminometer Mithras LB 940 (Figure 1). Bioluminescence of the reporters was simultaneously triggered by single injection of Ca2+ solution and discriminated using bioluminescent signal spectral and time resolution: during the first second photometer registered violet signal transmitted through optical filter I; the next 0.3 s go for the replacement for filter II; green light is registered for the last 5 s. Figure 1. Bioluminescent signals of obelin mutants W92F‐H22E (gray) and Y138F (dark gray), transmitted through filter I – 1) and filter II – 2); signals of obelins mixture, transmitted through filter I (during the first s) and then through filter II – 3). Dash line shows the time for filters change. The approach was tested on the solid‐phase assay of two pairs of targets in a sample: two gonadotropic hormones and two prolactin forms (total and IgG‐bound). Recombinant photoproteins were obtained and their conjugates with corresponding immunoglobulines were synthesized according to methods, described in (2). Follicle‐stimulating hormone (FSH) and luteinizing (LH) hormone were quantified by calibration curves, obtained on the base of standard sera. The results of bioluminescent analysis correlated well with the calculated values. Total prolactin was quantified with the help of calibration curve, obtained on the base of standard serum. The results of bioluminescent analysis correlated well with RIA data (R2 = 0.87, N = 117). For quantitative determination of macroprolactin in clinical samples we offered a model analytical system, which reflects the processes taking place in serum at immunological complexes formation. Consequently, we found the dependence of green reporter signal upon human immunoglobulin concentration, which we used as the calibration curve for macroprolactin detection. The results of the developed assay correlated well with those of RIA and were confirmed by gel‐chromatography data. Acknowledgements This work was supported by grant №76 of the Russian Academy of Sciences, Siberian Branch and by the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant № 11. G34.31.058) References Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES. Anal. Bioanal. Chem. 2008;391:2891–6. Frank LA, Petunin AI, Vysotski ES. Anal. Biochem. 2004;305:240–6. Novel Single Step Dual Luciferase Reporter Gene Assays Using Spectral Resolution Jorma Lampinena, Jae Choib, Megan Dobbsb, Doug Hughesb, Janaki Naraharib and Brian Webbb aThermo Fisher Scientific, Sample Preparation and Analysis, Vantaa, Finland bThermo Fisher Scientific, Pierce Protein Research, Rockford IL, USA Luciferase genes are the most common reporter genes used to study gene regulation. The most commonly used luciferase enzymes are the firefly luciferase and Renilla luciferase. The light emission spectra of the firefly and Renilla luciferases are rather wide and the peaks are located at 562 nm and 486 nm respectively. This makes it quite difficult to separate these two luciferase emissions based on wavelength. Therefore, dual luciferase assays are commonly performed as two‐step assays where the light reaction of one luciferase is quenched before another light reaction is initiated. Thermo Scientific has developed a new luciferase reporter gene family and corresponding luciferase activity assays that use novel luciferase genes from Cypridina and Gaussia combined with red firefly luciferase gene and green shifted Renilla gene. These new luciferases produce light emission spectra that are so widely spread over the visible wavelength range that luciferase emissions can be easily separated using spectral discrimination. Cypridina luciferase has emission maximum at 463 nm, Gaussia at 485 nm; green shifted Renilla has an emission peak at 525 nm and red firefly at 610 nm. In addition, Gaussia and Cypridina luciferases are secreted making it possible to measure luciferase activity without complicated cell lysis. This presentation explains the basic features of these luciferase constructs and performance of these new dual luciferase assays. A simple single step luciferase assay protocol was developed using spectral resolution. It makes the assay easy to perform and simple to automate for high throughput analysis. Spectral resolution is performed by either using spectral scanning luminometers or using a normal microplate luminometer that is equipped with specially designed high transmission filters optimized for these assays. The presentation focuses on clear benefits of these novel luciferase reported genes assays: very high brightness giving high sensitivity and reproducibility, easy non‐destructive analysis due to the secretion and simple detection of multiple luciferase activities. Biotinylated in vivo obelin produced in E. coli cells MD Larionovab, SV Markovaa,b, LA Franka,b and ES Vysotskia,b aPhotobiology Lab, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia bSiberian Federal University, Krasnoyarsk 660041, Russia E‐mail: larionova.marina@inbox.ru Ca2+‐regulated photoprotein obelin derivatives including its biotinylated forms have been shown to hold much promise as bioluminescent labels for binding assays (1). Chemical biotinylation of obelin requires several steps to prepare a label and leads to loss of bioluminescent activity (up to 30%), as well as to heterogeneity of biotinylated samples. In this study we describe highly effective production of site‐specific in vivo biotinylated obelin in E. coli cells. Applying the pET expression system, we succeeded in obelin synthesis amounting up to 70% of total cellular protein in E. coli cells (2). To produce biotinylated obelin, a short artificial biotin acceptor peptide which is effective substrate mimic for E. coli biotin ligase (BirA) (3) was genetically fused with obelin N‐terminus. Under appropriate growth conditions the tagged obelin synthesized from this expression construction was biotinylated. But biotinylated fraction of synthesized photoprotein was only 10‐15%, most likely due to the limited capacity of cellular BirA to biotinylate the overexpressing obelin. To overcome this limitation we used simultaneous expression of the BirA biotin ligase and obelin from one vector. The birA gene amplified from E. coli genome by PCR was inserted behind obelin gene through ATGA stop/start overlapping – translational coupling used for birA translation in E. coli genome. To improve expression properties of the obtained strain, the birA gene was cloned in the truncated form with deletion of the DNA‐binding domain acting as biotin operon repressor. Despite localization in the insoluble fraction of E. coli cells, the modified obelin was found to be biotinylated in vivo with high efficiency (~90%). Highly pure biotinylated apoobelin was obtained from inclusion bodies and converted in an active photoprotein at incubation with coelenterazine. The in vivo biotinylated obelin was successfully tested in a model immunoassay. This work was supported by the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). References Vysotski ES, Markova SV, Frank LA. Calcium‐regulated photoproteins of marine coelenterates. Molecular biology 2006;40(3):355–67. Markova SV, Vysotski ES, Lee J. Obelin hyperexpression in E. coli, purification and characterization. Bioluminescence & Chemiluminescence. World Scientific Publishing Co. Pte. Ltd. 2001;115–8. Shatz PJ. Use of peptide libraries to map the substrate specifity of a peptide modifying enzyme: a 13 residue consensus peptides biotinylation in Escherichia coli. Biotechnology 1993;11:1138–43. Application of immunoluminescence for tumor cell immunophenotyping and functional analysis of cell surface proteins Friedemann Laube Institute of Physiological Chemistry, Martin‐Luther‐University Halle‐Wittenberg, Halle, Germany Due to their accessibility, receptors and other cell surface proteins of the plasma membrane constitute the main targets for protein‐based drugs. Therefore, the targeting of tumor‐associated components by monoclonal antibodies (mAb) or small inhibiting molecules is a current therapeutic concept. Cell surface proteins may vary during the multistage process of metastasis and can confer critical capabilities on tumor cells. The immunoluminescent technique utilized secondary antibodies labelled with horseradish peroxidase (HRP). To detect the immunological response, the HRP‐catalyzed reaction of luminol with hydrogen peroxide including p‐iodophenol as enhancer was used. Reaction conditions were set to release luminescence at pH 7,4 and substrate concentrations were according to Kricka and Thorpe(1). In addition to the previous detection of different tumor‐associated proteins(2) cells of the human melanoma cell line IGR‐1 were characterized by melanotransferrin (MTf) and transferrin receptor‐1 (TfR‐1, CD71). While the function in iron transport of the TfR‐1 is well documented the functional importance of MTf is not yet fully understood. However, there are some recent insights into MTf function concerning tumorigenesis in melanoma. Both cell surface proteins were detected by the immunoluminescent technique using three different polyclonal antibodies for MTf and two mAb for the TfR. As an unexpected result, MTf was found to be resistant to phosphatidylinositol‐specific phospholipase C. However, MTf as well as TfR were sensitive to proteolytic degradation by pronase E and trypsin. As already shown for the upregulation of M6P/IGF‐II receptor(2) and alkaline phosphatase(3) by mannose‐6‐phosphate (M6P), this agent also stimulated the MTf upregulation. Obviously, M6P may cause signalling processes via the M6P/IGF‐II receptor stimulating the expression of multiple proteins. In contrast to MTf the TfR was upregulated by hyaluronic acid (HA) in a concentration dependent manner. This result suggests a possible interaction of HA with membrane‐bound CD44. Previously, CD44 was detected as the main receptor for HA on IGR‐1 cells(4). These results suggest that the signal transduction of HA‐CD44 might be linked with the TfR upregulation according to the sequence: HA – CD44 – ErbB2 – N‐WASP – β‐Catenin – c‐Myc – TfR. The TfR was identified as one target gene of the transcription factor c‐Myc(5). Recombinant human IGF‐II failed to stimulate the upregulation of MTf and TfR but increased the M6P/IGF‐II receptor expression. This approach provides some insights into the complex interplay of diverse cell surface receptors. Certain components of this network may represent possible targets for the tumor suppression. References Kricka LJ, Thorpe GHG. Bioluminescent and chemiluminescent detection of horseradish peroxidase labels in ligand binder assays. In: Van Dyke K., Van Dyke R. (Eds.) Luminescence immunoassay and molecular applications. Boca Raton, CRC Press, 1990;77–98. Laube, F. Mannose‐6‐phosphate/insulin‐like growth factor‐II receptor in human melanoma cells: effect of ligands and antibodies on the receptor expression. Anticancer Res. 2009;29:1383–8. Ishibe M, Rosier RN, Puzas JE. Activation of osteoblast insulin‐like growth factor‐II/cation‐independent mannose‐6‐phosphate receptors by specific phosphorylated sugars and antibodies induce insulin‐like growth factor‐II effects. Endocrine Res. 1991;17:357–66. Laube F. Co‐localization of CD44 and urokinase‐type plasminogen activator on the surface of human melanoma cells. Anticancer Res. 2000;20:5045–8. O'Donnell KA, Yu D, Zeller KI, Kim J‐W, et al. Activation of transferrin receptor 1 by c‐Myc enhances cellular proliferation and tumorigenesis. Mol. Cell. Biol. 2006;26:2373–86. Theoretical Studies on Dynamics, Mechanism and Active Species in Luminescence and Photoconversion of Chromophores in Protein Xin Li,a Lung Wa Chung,a Lina Ding,a and Keiji Morokumaa,b,* aFukui Institute for Fundamental Chemistry, Kyoto University, Kyoto 606‐8103, Japan bCherry L. Emerson Center for Scientific Computation and Department of Chemistry, Emory University, Atlanta, GA 30322, USA Photobiological systems are attracting a lot of attention. For instance, photoactivatable fluorescent proteins have recently been developed for new bio‐imaging technologies and can be categorized into three types (Figure 1): irreversible photoactivation via decarboxylation, irreversible photoconversion via cleavage of the protein backbone, and reversible photoswitching between fluorescent and non‐fluorescent states via photoisomerization. High‐level QM and ONIOM(QM:MM) calculations have been performed to investigate reaction mechanisms of the reversible photoswitching in Dronpa,1,3 the irreversible green‐to‐red photoconversion in Kaede,2 and possible decarboxylation mechanism in PA‐GFP. New mechanisms involving photoisomerization coupled with excited‐state proton transfer for the photoactivation in Dronpa and competitive E1cb pathway for Kaede were found.1–3 In addition, the primary event of photodynamics of Dronpa was further elucidated by non‐adiabatic (NA) ONIOM molecular dynamics (MD) simulations,3 showing distinctive photodynamics for the chromophore in different protonation states and/or protein structures. Moreover, photodynamics of all‐trans retinal protonated Schiff base in bacteriorhodopsin (bR) as well as in a solution was also explored by NA ONIOM MD simulations (Figure 2).4 The protein matrix in bR was found to promote the bond‐specific, unidirectional and ultrafast photoisomerization with a high quantum yield.4 In addition, the possible reaction mechanism of an efficient firefly bioluminescence will be discussed.5,6 Figure 1. Photo‐activation mechanisms in three types of PAFPs. Figure 2. (a) ONIOM‐optimized active‐site structure of bR. (b) Excited‐state population for photoisomerization of the all‐trans retinal in bR and methanol by ONIOM MD simulations. References Li X, Chung LW, Mizuno H, Miyawaki A, Morokuma K. A Theoretical Study on the Natures of the On‐ and Off‐States of Reversibly Photoswitching Fluorescent Protein Dronpa: Absorption, Emission, Protonation and Raman, J. Phys. Chem. B 2010;114:1114–26. Li X, Chung LW, Miyawaki A, Morokuma K. Competitive Mechanistic Pathways for Green‐to‐Red Photoconversion in the Fluorescent Protein Kaede: A Computational Study, J. Phys. Chem. B 2010;114:16666–75. Li X, Chung LW, Miyawaki A, Morokuma K. Primary Events of Photodynamics in Reversibly Photoswitching Fluorescent Protein Dronpa, J. Phys. Chem. Lett. 2010;1:3328–33. Li X, Chung LW, Morokuma K. Photodynamics of All‐trans Retinal Protonated Schiff Base in Bacteriorhodopsin and Methanol Solution, J. Chem. Theo. Comp. 2011;7:2694–8. Chung LW, Hayashi S, Lundberg M, Nakatsu T, Kato H, Morokuma K. Mechanism of Efficient Firefly Bioluminescence via Adiabatic Transition State and Seam of Sloped Conical Intersection, J. Am. Chem. Soc. 2008;130:12880–1. Li X, Chung LW, Morokuma K. Modeling Photobiology Using Quantum Mechanics (QM) and Quantum Mechanics/Molecular Mechanics (QM/MM) Calculations, in “Computational Methods for Large Systems: Electronic Structure Approaches for Biotechnology and Nanotechnology”, ed. J. R. Reimers, John Wiley, Hoboken, NJ, 2011;397–434. Conjugation of Luciola mingrelica firefly luciferase with biospecific proteins through the enzyme SH‐groups GY Lomakina and NN Ugarova Lomonosov Moscow State University, Moscow, Russia E‐mail: lomakinagalina@yahoo.com Firefly luciferase is a promising enzyme label for bioluminescent‐based assays. However the attempts to prepare the functional active and stable covalent conjugates of luciferase through the surface active NH2‐ or SH‐groups of amino acid residues with biospecific molecules such as antigen, antibody and nucleic acid have largely been unsuccessful. It is believed that lysine and cysteine residues are important for luciferase activity and chemical modification of native surface residues results in loss of the enzyme activity. The feature of Lиciola mingrelica firefly luciferase is availability of five active SH‐groups of non‐conservative cysteine residues. Three of them are located on or near enzyme surface with exposed to solvent SH‐groups. The distance from cysteines to active site and from each other more than 30Å and they do not affect the catalysis. In this work the method of conjugation of Lиciola mingrelica firefly luciferase with biospecific proteins through the free thiol groups of the enzyme and NH2‐groups of protein using heterobifunctional cross‐linker N‐succinimidyl 3‐(2‐pyridylditio)‐propionate (SPDP) was developed. The active conjugates luciferase‐bovine serum albumin (4TS‐BSA) and luciferase‐chicken egg avidin (4TS‐Avi) were obtained for the first time by the scheme (Fig. 1). Figure 1. Scheme of firefly luciferase conjugates synthesis. Two forms of L.mingrelica firefly luciferases with C‐terminal His6‐tag were used for conjugation: recomdinant wild type enzyme with three surface SH‐groups and thermostable luciferase mutant, 4TS, with two surface SH‐groups (the surface Cys146 residue was mutated) (1). Conjugates were purified by metal chelate chromatography. The yield of luciferase activity of conjugates was 60–70% of that of initial enzyme. The amount of non‐modified enzyme was less than 1%. The time of the conjugation depended on the SH‐group quantity: the reaction time for mutant without of surface Cys146 residue increased in 20‐ fold. The conjugation resulted in improvement of the kinetic properties (КmATP was 4‐times less for BSA‐Luc, and КmLH2 – 5 times for Avi‐Luc) and increase of the stability in comparison with the initial enzyme. The conditions of the low enzyme concentrations detection were optimized. The luciferase detection limit was 10−13 mol/L. The conjugate BSA‐Luc was successfully used in the competitive enzyme immunoassay for the quantitative detection of albumin in concentration range: 5–300 µg/mL. The Salmonella typhimurium cells assay was developed using biotinylated antibodies and conjugate Avi‐Luc. Reference 1. Koksharov M, Ugarova N. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein engineering, design and selection. 2011;24:835–44. Characteristics of a coupled enzymatic system of luminous bacteria co‐immobilized with substrates and stabilizers into starch gel Victoria Lonshakovaa, Elena Esimbekovab,a and Valentina Kratasyuka,b aSiberian Federal University, Krasnoyarsk, 660041, Russia E‐mail: VKratasyuk@gmail.com bInstitute of Biophysics SB RAS, Krasnoyarsk, 660036, Russia Enzymes isolated from luminescent bacteria are extensively used for environmental monitoring (1). One of the most promising reagents is a disk‐shaped reagent based on the bacterial enzymes NADH:FMN‐oxidoreductase and luciferase, co‐immobilized into a starch or gelatin gel with their substrates NADH and myristic aldehyde (2). But the reagent can lose up to 80% of its activity after storage at 4°C for 1 year. However, the entrapment of special additives into the reagent can lead to the stabilization of enzymes, thus improving reagent characteristics. The goal of this work was to develop an immobilized multicomponent reagent with high activity, long storage time and high sensitivity to toxic substances. This reagent includes the coupled enzymatic system NADH: FMN‐oxidoreductase‐luciferase, their substrates and enzyme stabilizers. The immobilized reagent was a dried film forming a disc with a diameter of 6–7 mm; its dry weight was 1.5 ± 0.2 mg. We varied the concentration of the set of enzyme stabilizers inside the reagent: dithiothreitol (DTT), bovine serum albumin (BSA) and mercaptoethanol It was shown that the stabilizers increased the luminescence intensity of the immobilized reagent – BSA by 900% and DTT or mercaptoethanol by 200%, compared to the activity of the reagents without any stabilizers. The residual activity of reagents stored at 4°C for 6 months was examined. The stabilizing effect was observed when immobilized reagents included BSA or DTT. Mercaptoethanol had no stabilizing effect. The stabilizers reduced the sensitivity of the immobilized reagent to model toxicants (heavy metal salts, quinones and phenols). An exception was the reagent that included 0.1 mM DTT (Table 1), when the residual intensity of luminescence in the presence of toxic substances did not differ from the control measurements of the reagent without stabilizers. Table 1. Effects of some organic pollutants on the bioluminescence of a coupled enzyme system co‐immobilized into a starch gel with their substrates and 0.1 mM DTT Class Substance MPS, mg/L EC50, mg/l Control DTT Heavy metal salts CuSO4 1 10 16 CrCl2 0,05 479 586 HgCl2 1 0,62 0,73 Phenols Pyrocatehin 0,1 84,8 148,4 Hydroqinone 0,2 3,06 4,72 Quinones Benzoquinone 0,1 0,002 0,003 Tolyquinone ‐ 0,00008 0,00008 Timoquinone ‐ 0,00005 0,0007 Naphtoquinone 0,25 0,001 0,0016MPS – maximum permissible concentration EC50 ‐ concentration of the active substance when bioluminescence is inhibited by 50 %. Thus, the best result was achieved when 0.1 mM DTT was used as a stabilizer. In this case, the immobilized multicomponent reagent combined high sensitivity to toxic substances with a longer period of reagent storage. This work was supported by the Federal Agency of Science and Innovations (contract No 02.740.11.0766), the Russian Academy of Sciences (Program “Molecular and cellular biology”, grant No 6.2), President of RF (grant Leading scientific school No 64987.2010.4), the Program of the Government of the Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). References Vetrova E, Esimbekova E, Remmel N, et al. A bioluminescent signal system: detection of chemical toxicants in water. Luminescence 2007;22(3):206–14. Patent of Russian Federation N 2252963. Method for production of immobilized multi‐component reagent for bioluminescent analysis. Computational Investigation of the Photinus pyralis Luciferase‐Oxyluciferin System Luís Pinto da Silva* and Joaquim CG. Esteves da Silva Centro de Investigação em Química (CIQ‐UP), Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre 687, 4169‐007 Porto (Portugal) E‐mail: luist311@hotmail.com Bioluminescence is a light‐emitting phenomenon that occurs in living organism, in which an excited state molecule is produced in a luciferase catalyzed reaction. The color of bioluminescence emitted by different firefly species range from 530 to 640 nm, despite the fact that oxyluciferin is the sole light emitter.1 Thus, the main factor in the color tuning mechanism must be the interactions made between oxyluciferin and the different luciferases. We have employed various computational methodologies to study for the first time the bioluminescence of an experimentally‐obtained crystal structure of Photinus pyralis luciferase.2 The interaction of excited state oxyluciferin with active site molecules was studied at the TD‐PBE0/6‐31+G(d) level of theory, while molecular mechanics and dynamics were used to optimize the luciferase‐oxyluciferin complex.3 The emission of oxyluciferin is affected the most by ionic interaction with AMP (blue‐shift), π‐π stacking with Phe247 (red‐shift) and by hydrogen‐bonding with neighboring water molecules (red/blue‐shift), as can be seen in Figure 1. Arg218 and His245 are also relevant molecules in the color tuning mechanism. For the contrary, amino‐acids as Ile351, Thr251 and Ala348 do not have any contribution to the color of bioluminescence, thus being possible targets for mutations with the objective of tuning the emission. The results here presented are consistent with the ones referring to other firefly species, indicating that we have defined a mechanism of color tuning that may be applied in all firefly species.3,4 Figure 1. Graphic representation of the contribution of active site molecules to the color of light emitted by oxyluciferin. References Pinto da Silva L, Esteves da Silva JCG. Computational Studies of the Luciferase Light‐Emitting Product: Oxyluciferin. J. Chem. Theory Comput. 2011;7:808–17. Auld DS, Lovell S, Thorne N, Lea WA, Maloney DJ, Shen M, Rai G, Battaile KP, Thomas CJ, Simeonov A, Hanzlik RP, Inglese J. Molecular Basis for the high‐affinity binding and stabilization of firefly luciferase by PTC124. Proc. Nat. Acad. Sci. USA 2010;107:4878–83. Pinto da Silva L, Esteves da Silva JCG. TD‐DFT/Molecular Mechanics Study of the Photinus pyralis Bioluminescence System. Submitted 2011. Pinto da Silva L, Esteves da Silva JCG. Study of the Effects of Intermolecular Interactions on Firefly Multicolor Bioluminescence. Chemphyschem 2011;12:3002–8. Comparison of purity and activity of D‐luciferin from 8 manufacturers Arne Lundin*, Björn Malm and Nadia Touma BioThema AB, Handens stationsväg 17, 13640 Handen, Sweden *E‐mail: arne.lundin@biothema.com Impurities in D‐luciferin may be strongly inhibitory in the luciferase reaction. For example a 4 % addition of L‐luciferin in D‐luciferin decreases the activity to 50% (1). Using D‐cysteine contaminated by L‐cysteine in the last step of the synthesis will give a low luciferase activity. D‐luciferin is sensitive to light and oxygen, and dehydroluciferin is a potent inhibitor. When dissolving the D‐luciferin a high pH will result in a rapid racemization. In assays of ATP or luciferase, inhibitors will give a decreased sensitivity and an increased detection limit. Correct results may still be obtained provided each assay is calibrated by an internal standard. Assays of ATP may be calibrated using a certified, liquid‐stable ATP standards (2). In assays of luciferase the situation is somewhat more complicated as several different luciferases are available, both native and recombinant, and the correct type of standard must be chosen. BioThema produces a range of kits both for ATP assays and for luciferase assays. We have since the 90s evaluated D‐luciferin from various manufacturers to make sure that we use the highest quality. The evaluation relied on a lyophilized in‐house ATP reagent similar to our 11‐501 ATP Reagent SL (Stable Light) but without D‐luciferin. The light emission from the D‐luciferin preparation under evaluation was calculated as a percentage of what we obtained with a “gold standard” luciferin from the manufacturer producing the highest light emission, i.e. the highest quality. The activity was compared at 100 (below optimal), 200 (optimal) and 300 (above optimal) µg/mL. At the optimal D‐luciferin level, activities from different manufacturers in earlier measurements ranged from close to 100% to as low as 30%. In 2010 BioThema took over the production of D‐luciferin from our previous supplier. We run the synthesis in a newly built laboratory specifically designed for the purpose, e.g. HEPA filters on the inlet air. We make sure that we still produce the same high quality as the previous supplier by comparing to the original gold standard and to samples obtained from other manufacturers. In addition to the biochemical performance test using the in‐house ATP reagent mentioned above we also run HPLC using both a chiral AGP column and a RP C8 column using a UV detector (225 nm) and a fluorescence detector (excitation 330 nm and emission 550 nm). The most important QC parameter is biochemical performance. In Table 1 the preparations have been arranged in descending activity at 200 µg/mL. There is no obvious correlation between biochemical performance and HPLC purity. The latter therefore should always be complimented with biochemical performance testing. Table 1. Optical and chemical purity as measured by HPLC and luciferase activity as measured by an in‐house kit for D‐luciferin. All numbers are in percent. The activities at 100 and 300 µg/mL are expressed in percent of the activity at 200 µg/mL. The activity at 200 µg/mL is expressed in percent of the gold standard and is the best measure of quality Supplier Chiral column UV detector Chiral column Fluorescence detector RP C8 column UV detector RP C8 column Fluorescence detector Biochemical performance D‐luciferin L‐luciferin Others D‐luciferin L‐luciferin luciferin Others luciferin Others 200 µg/mL 100 µg/mL 300 µg/mL BioThema 99.8 0.2 0.0 99.8 0.2 100.0 0.0 100.0 0.0 101.3 90.9 96.9 2 99.6 0.1 0.4 100.0 0.0 99.5 0.5 99.1 0.9 98.9 90.6 97.9 3 98.4 0.2 1.4 99.7 0.3 99.6 0.4 99.9 0.1 98.7 87.8 100.1 4 98.0 0.3 1.7 99.6 0.4 100.0 0.0 100.0 0.0 98.2 91.9 97.6 5 99.6 0.1 0.3 99.9 0.1 100.0 0.0 100.0 0.0 94.9 89.5 99.4 6 98.9 0.2 0.9 99.7 0.3 99.5 0.5 99.4 0.6 93.7 92.4 97.5 7 99.6 0.2 0.2 99.8 0.2 99.7 0.3 99.9 0.1 92.1 90.8 94.9 8 99.1 0.3 0.5 99.7 0.3 99.9 0.1 99.9 0.1 76.7 94.5 96.4 Minimum 98.0 0.1 0.0 99.6 0.0 99.5 0.0 99.1 0.0 76.7 87.8 94.9 Maximum 99.8 0.3 1.7 100.0 0.4 100.0 0.5 100.0 0.9 101.3 94.5 100.1 References Lundin A. Applications of firefly luciferase. In Luminescent Assays: Perspectives in Endocrinology and Clinical Chemistry (Eds. Mario Serio, Mario Pazzagli), Raven Press, 1982;29–45. Lundin A, Inglis R, Touma N. Certified ATP Standard. Luminescence 2010;25:159–61. Color tuning of bioluminescence reaction by modifying the hydrogen bond network around the active site in firefly luciferase Mika Maenaka, Dai‐ichiro Kato, Takaya Kubo, Kazuki Niwa, Yoshihiro Ohmiya, Masahiro Takeo and Seiji Negoro University of Hyogo, 2167 Shosha, Himeji, Hyogo, 671‐2280 Japan E‐mail: maenaka_maenaka@yahoo.co.jp Nowadays bioluminescence of firefly is finding increasing use as bioimaging tools. The bioluminescent reaction is achieved by the chemical conversion of D‐luciferin to oxyluciferin in the active site of firefly luciferase. Although the light emitter is unique, the range of emission is diverse from green to red (530–640 nm) by the difference of luciferase origins. Moreover, even if the same luciferase were used, the emission color is also shifted by changing the reaction conditions such as pH and/or temperature. In spite of many efforts to clarify the color changing mechanism, we can't settle the dispute to agreeable theories. This is partially due to the lack of comprehensive data of amino acid positions which have effect on the emission color. Among many proposed hypotheses of color modulation mechanism, two theories, “rigidity of the active site(1)” and “strength of covalent character of phenolic hydroxide on benzothiazole ring of oxyluciferin(2)” are dominant to date to explain the emission color derivation (Fig. 1). Though there are many examples to support former hypothesis by using mutagenesis and/or structural analysis, there is no direct evidence to explain later one. Thus, we have prepared the mutant libraries related to the “covalent character” and carried out the detailed characterization of each one. Figure 1. Two proposed mechanism of bioluminescence color determination in firefly lusiferase. As “the strength of covalent character” would be affected by the changing of hydrogen bond network between the phenolic hydroxide on benzothiazole ring and amino acid residues around the active center, we predicted the candidate residues to influence the hydrogen bond networks by using the molecular dynamics (MD) calculation method and finally conclude to focus on two positions (S286 and V239). Based on this information, 19 single amino‐acid mutants for each position were constructed by point mutagenesis technique, which resulted in obtaining the mutants emitted various colors (562–604 nm). These positions also showed a marked effect on the pH sensitivity and quantum yield. These amino acids locate on far from the substrate ( > 8 Å). By comparing the predicted optimized structure of these derivatives, the hydrogen bond networks around the phenolic hydroxide were changed dramatically despite very little conformational change of amino acid side chains around the substrate (Fig. 2). We conclude that the MD simulation data provides the fundamental information to select the ideal mutational position for tuning the emission color. Figure 2. The states of hydrogen bond network around the phenolic hydroxide. (gray: WT green: V239E red: V239Q). References Nakastu T, Ichiyama S, Hiratake J, Saldanha A, Kobayashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence, Nature 2005;440:372–736. Hirano T, Hasumi Y, Ohtsuka K, Maki S, Niwa H, Yamaji M, Hashizume D, Spectroscopic studies of the light‐color modulation mechanism of firefly (beetle) bioluminescence, J. Am. Chem. Soc. 2009;131:2385–96. News in Echinoderm's luminescence J Mallefet (Marine Biology Laboratory, UCL, Belgium) Stating that there are many bioluminescent organisms in the marine environment is a commonplace (1), however certain marine phyla remain poorly understood. In Echinoderms, luminescence occurs in four of the five classes: Crinoidea, Holothuroidea, Asteroidea and Ophiuroidea (2). Until recently, in many cases the information was solely descriptive and limited to morphological, ecological and some features of bioluminescence. One major reason of this poorly documented phenomenon in echinoderms is restricted accessibility that limits the number of species studied (3). For the last ten years, a series of field trips and participation to one deep‐sea cruise allowed to discover and describe the luminous capabilities of numerous ophiuroids as well as some holothuroids and one crinoid species (Table 1). Within the echinoderm phylum, bioluminescence is not uniformly distributed across the classes: ophiuroids (66 species) and holothurians (31 species) represent 80 % of luminous echinoderm species while asteroids (20 species) and crinoids (3 species) contribute to 17 and 3% respectively (Fig 1) . A multidisciplinary approach allowed to obtain physiological, morphological, ecological, ethological and finally biochemical data for some ophiuroid species mainly because luminous representatives of this class are present from the intertidal zone downwards. This presentation attempts to synthesize recent information mainly on ophiuroids luminescence but will also present literature data on luminescence observations in other echinoderm's classes in order to illustrate the diversity of this amazing phenomenom (4) (5) (6). New field surveys must be performed to increase the number of echinoderm species tested to highlight a possible link between luminescence and phylogenetic distribution of this capability in order to understand why so many echinoderms glow in the dark. Figure 1. Distribution of luminous capabilities in echinoderms. Table 1. Updated number of species in each echinoderm class and the known luminous species, adapted from (2) to (6) Classes Species number Total Luminous 1995 2011 Echinoidea 838 0 0 Ophiuroidea 2278 33 66 Holothuroidea 1430 30 31 Asteroidea 1745 20 20 Crinoidea 576 3 4 This work was supported by the financial support ASSEMBLE grant agreement no. 227799 and FNRS research grant 1.5.278.08; J. Mallefet is a research associate of the FNRS. Contribution to the Biodiversity Research Center (BDIV) and to the Centre Interuniversitaire de Biologie Marine (CIBIM). References Hastings JW. Bioluminescence. In Cell Physiology Source Book, ACA press, San Diego, 1995;665–81. Herring PJ. Bioluminescent echinoderms: Unity of function in diversity of expression? Echinoderm Research 1995;9–17. Mallefet J. Echinoderm bioluminescence. In Bioluminescence in Focus – A Collection of Illuminating Essays (Eds.Victor Benno Meyer‐Rochow), Research Signpost, India, 2009;67–83. Harvey EN. Bioluminescence. Academic Press, 1952, New York. Herring PJ. Systematic distribution of bioluminescence in living organisms. J. Biolum. Chemilum 1987;1:147–63. Herring PJ. New observations on the bioluminescence of echinoderms. J. Zool. London 1974;172:401–18. High‐active truncated luciferases of copepod Metridia longa and their characterization as secreted reporters in mammalian cells Svetlana V. Markovaa,b, Ludmila P. Burakova a,b and Eugene S. Vysotskia,b aPhotobiology Lab, Institute of Biophysics SB RAS, Krasnoyarsk 660036, Russia bSiberian Federal University, Krasnoyarsk 660041, Russia E‐mail: smarkova@mail.ru Bioluminescent reporters are sensitive and convenient tool for real‐time monitoring of biological processes in living cells. The secreted reporters enable continuous monitoring of intracellular events without destroying cells or tissues. Metridia luciferase (MLuc) from the copepod Metridia longa, a small (~22 kDa) coelenterazine‐dependent luciferase containing a natural signal peptide for secretion, was effectively applied as a secreted bioluminescent reporter in mammalian cells (1). To improve bioluminescent properties of MLuc reporter we obtained the high‐active MLuc mutants by deletions of the N‐terminal variable part of amino acid sequence. The three MLuc mutants truncated at Met54, Thr73, and Met80 including signal peptide with calculated molecular mass 17.9, 16.0, and 15.1 kDa, respectively, were constructed. For functional analysis the MLuc variants were produced in E. coli cells, purified and refolded to an active protein from inclusion bodies. We demonstrate that the truncated MLuc mutants have significantly increased light intensity and efficiency of bioluminescent reaction as against the wild type enzyme but substantially retain other properties. Under exactly the same measurement conditions (substrate and luciferase concentrations, pH, temperature, buffer) the truncated variants produce higher intensities but decay faster than the wild type enzyme, so that the total bioluminescence yield is about 2.4 and 4.8 times higher for 17.9 and 16.0 kDa variants. The high‐active truncated variants of MLuc with 17.9 and 16.0 kDa displaying ~6 and ~10‐fold increase of maximal bioluminescent activity were transiently expressed in HEK293 cells. The results clearly suggest that the truncated Metridia luciferases are well suited as secreted reporters ensuring higher detection sensitivity in comparison with a wild type enzyme. This work was supported by the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). Reference 1. Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES. Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme. J. Biol. Chem. 2004;279:3212–7. How to analyse bioluminescence time series from in situ observatories? Example from high frequency records and real time data at the ANTARES site S Martinia, D Nerinia and C Tamburinia a Mediterranean Institute of Oceanography, (MIO, COM), Marseille France Introduction Sampling, quantifying and observing marine bioluminescence in the natural environment is currently done using manned submersibles, autonomous underwater vehicles and underwater photometers. Observations of deep‐sea bioluminescence are mainly based on mechanical stimulation using for instance pumped flow through a turbulence‐generating grid or a downward moving grid knocking on the organisms. The recent use of in situ sensor technology and additional observation platform such as autonomous underwater vehicles and undersea observatories to see the bioluminescence provides new insights. These technologies provide an understanding of the dynamics and the ecological importance of bioluminescent organisms during long periods that have to be combined with more detailed genomic and physiologic studies in the laboratory (Widder, 2010). In this study, we use data from the ANTARES neutrino telescope, located 40 km off the French Mediterranean coast at 2475 m depth. This structure is mainly dedicated to the search of the Cherenkov light emission radiated by elementary charged particles named muons that are produced by neutrinos interactions. Moreover, this deep observatory also provides real‐time data of in situ bioluminescence, at high frequency, coupled with oceanographic data sampled at the same time (Tamburini et al., submitted). Data analysis methods In natural environments, non‐stationary and non‐linearly datasets are commonly recorded but only two methods are existing to explore these particular data. The wavelet (Torrence and Compo, 1998) and the Hilbert‐Huang (Huang et al., 1998) methods are dedicated to analyse fluctuations at various scales of time and frequencies. Both methods decompose time series into bases of functions dedicated to specific frequencies. In this study, Martini et al. (in prep.) provide an analysis of time series using these two decomposition methods. Several long time series between the end of 2007 and the middle of 2010 have been analysed providing informations on links between the biological variable bioluminescence and oceanographic variables. These relations are characterized using common frequencies excited in the signals and time where they are excited. Bioluminescence as bio‐indicator of changes Bioluminescence intensity varies during the time series but two distinct periods of about one month in 2009 and 2010 are clearly identified as very high bioluminescence activity at the ANTARES site (Figure 1). During the whole time series 2007‐2010, intense horizontal sea current speed is often linked to bioluminescence (coherency coefficient above 0.8) in a large range of frequencies. This relation is already well known as a physical stimulation of bioluminescent organisms. However during the two short high bioluminescence events the biological variable is closely related to salinity and also temperature (coherency coefficient above 0.8) at low frequency (period around 25 days). The phase coefficient during these periods is close to 0 meaning that bioluminescence and variables are varying in the same way (increasing or decreasing at the same time). These two events are related to changes in water masses in the deep sea close to the ANTARES station. Figure 1. Time and frequency spectra of bioluminescence decomposed with the wavelet method. High frequencies are excited along the whole time series and two high intensity bioluminescent events appear in 2009 and 2010 at low frequencies. Conclusion and perspectives These analyses are the first innovative step to propose the use of bioluminescence activity records by Eulerian observatories as a proxy of biological activity in the deep sea. As a working perspective, the determination of the part of micro or macro‐organisms involved in the high bioluminescent signal is crucial. Then, in a final aim bioluminescence sensors would be a new way to provide informations of ecological global changes taking place in the deep ecosystems. References Widder E. Bioluminescence in the Oceans: origins of biological, chemical and ecological diversity. Sciences 2010;328(5979):704–8. Huang NE, Shen Z, Long SR, Wu MC, Shih HH, Zheng Q, Yen NC, Tung CC, Liu HH. The empirical mode decomposition and the Hilbert spectrum for nonlinear and non‐stationary time series analysis. Proceedings of the Royal society 1998;454:903–95. Tamburini C, the ANTARES collaboration (submitted) Enhancement of deep‐sea pelagic activity by dense water formation. Nature Geoscience. Torrence C, Compo GP. A practical guide to wavelets analysis. Bulletin of the American meteorological society 1998;79(1):61–78 Martini S, Nerini D, Tamburini C. (in prep.) Comparison and complementarity in the Hilbert‐Huang and the wavelet methods: analysis of oceanographic non‐stationary and non‐linear long time series. Development of a triple color bioluminescent breast cancer cell line for high content analysis Laura Mezzanotte, Na An, Eric Kaijzel and Clemens Löwik Dept of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands The availability of multicolor luciferases expanded the potential of the development of cell based assays for high content analysis especially in the drug discovery and development process (1,2,3). Assays using cancer cell lines can give relevant biological information about compounds active in key pathways, their toxicity and their proapoptotic potential. Nowadays drugs acting on NFκB (nuclear factor kappa beta) signalling are largely investigated due to the involvement of the pathway in cancer progression (4). Here we report the establishment and validation of a triple color bioluminescent breast cancer cell line for the simultaneous monitoring of the NFkB signalling, cell viability and apoptosis. Briefly, the human breast cancer cell line MDA‐MB‐231 was transduced with a lentiviral vector for the expression of click beetle green luciferase CBG99 under the control of a constitutive promoter and selected with limit dilution method to create a stable MDA‐MB‐231 CBluc cell line. Subsequently, cells were transduced with a bicistronic bidirectional lentiviral vector (5) in which the PGK (phosphoglycerate kinase) promoter controls the expression of a transmembrane form of Gaussia luciferase. The vector was modified by the insertion of a cassette encoding for red PpyRE9 luciferase (6) under the control of a NFκB promoter. After transduction, cells were sorted using FACS (fluorescence activated cell sorting) and a polyclonal anti‐Gaussia luciferase antibody. In order to validate the stable triple color cell line, cells were plated in a black 96 well plate at a density of 5000/well, stimulated with 10ng/ml of TNFα and treated with different anticancer natural compounds (e.g. resveratrol, sulforaphane, gambogic acid, curcumin, celastrol and betulinic acid) that are known to act on NFκB signalling. Signals were collected from live cells using a luminograph (Ivis Spectrum) and a series of band pass filters (20nm). Information about cell viability and NFκB signallig can be evaluated at the same time by spectrally resolving the light emitted by green CBG99 and red PpyRE9 luciferase reporters, after addition of the single shared substrate D‐luciferin in live cells. Apoptosis was monitored by determination of caspase 3/7 activity. For this, DEVD‐luciferin, which can only be used as substrate after removal of the DEVD moiety by the caspase enzyme, was added to the cells. The cell line showed a 100 fold induction of NFκB promoter activity 24 hours after TNFα stimulation. By correcting the signals for cell viability (as shown in Figure 1 for resveratrol) the assay allowed to calculate the effect on NFκB signaling for all the compounds tested over a wide range of concentrations. The possibility of evaluating these processes at the same time, with no need to transfect with reporter plasmids, represents a great advantage in terms of predictability, time and cost. In addition, a xenograft mouse model can be established for monitoring the processes in vivo. Figure 1. Representative image of live MDA‐MB‐231 assay. Cells were treated with TNFa 10 ng/ml and increasing concentrations of resveratrol (1‐200 μM) for 24 hours. Non treated cells have been used as control.A) Unmixed image corresponding to green luciferase signals used to assess cell viability. B) Unmixed image corresponding to red luciferase signals used to monitor NFκB induction. C) Composite image representing the sum of the green and red signals. D) NFκB promoter fold induction in response to TNFα and resveratrol. Treatment with resveratrol induces significant inhibition of NFκB induction (* P < 0.05, **P < 0.01, two‐tailed Student t‐test). References Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini B, Roda A. Spectral‐resolved gene technology for multiplexed bioluminescent cell‐based assays and high‐content screening. Analytical Chemistry Jan 1, 2008;80(1):260–7. Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A. Cell‐based assays: fuelling drug discovery. Anal Bioanal Chem. Sep 2010;398(1):227–38. Mezzanotte L, Que I, Kaijzel E, Branchini B, Roda A, Löwik C. Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase. PLoS One. Apr 22, 2011;6(4):e19277. Prasad S, Ravindran J, Aggarwal BB. NF‐kappaB and cancer: how intimate is this relationship. Mol Cell Biochem. Mar 2010;336(1‐2):25–37. Epub 2009 Oct 8. Review. Branchini BR, Ablamsky DM, Davis AL, Southworth TL, Butler B, Fan F, Jathoul AP, Pule. Red‐emitting luciferases for bioluminescence reporter and imaging applications. Anal Biochem. Jan 15, 2010;396(2):290–7. Na IK, Markley JC, Tsai JJ, Yim NL, Beattie BJ, Klose AD, Holland AM, Ghosh A, Rao UK, Stephan MT, Serganova I, Santos EB, Brentjens RJ, Blasberg RG, Sadelain M, van den Brink MR. Concurrent visualization of trafficking, expansion, and activation of T lymphocytes and T‐cell precursors in vivo. Blood. Sep 16, 2010;116(11):e18–25. WHOLE‐CELL BIOLUMINESCENT BIOSENSORS FOR ON‐SITE ANTIDOPING SCREENING E Michelinia,b, L Ceveninia,b*, C Canalia, L Ekströmc, J Schulzec , M Garlec, A Ranec and A Rodaa,b aDepartment of Pharmaceutical Sciences, University of Bologna, Italy bINBB, Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy cLaboratory Medicine, Division Clinical Pharmacology, Karolinska Institutet, Stockholm, Sweden E‐mail: elisa.michelini8@unibo.it Many areas, such as medical diagnostics and anti‐doping analysis, would benefit from devices that can perform a rapid and cost‐effective screening without the need for equipped laboratories. Thanks to their ability to exploit highly specific biomolecular recognition mechanisms integrated within the detection system, biosensors can satisfy many of the analytical requirements related to on‐site analysis. As a branch of biosensors, engineered bioluminescent (BL) cells exploiting BL reporter gene technology are now emerging as sensitive analytical tools.1 Yet the issue of long‐term cell viability preservation and their integration in robust and portable device remains unsolved.2 Conventional testosterone doping tests are based on the urinary testosterone/epitestosterone glucuronides ratio (T/E) determination, usually performed by GC/MS. About forty percent of the individuals devoid of the UGT2B17 gene, encoding for the major enzyme responsible for testosterone glucuronidation, never reach the cut‐off value even after the injection of 500 mg testosterone enanthate.3 We investigated whether a BL androgen‐responsive yeast strain could be used as a rapid cost‐effective anti‐doping screening tool. Cells were genetically engineered to express the human androgen receptor (hAR) which drives the expression of P. pyralis wild‐type luciferase (PpyWT, λmax = 557 nm) through the regulation of the androgen responsive element (ARE) in presence of hAR agonists; an internal viability control relying on constitutive expression of the P. pyralis red‐emitting mutant thermostable luciferase (PpyRE8, λmax = 618 nm) has been also introduced.4 Plasma and urine of healthy volunteers who were given 500 mg of testosterone enanthate i.m. were analyzed with the whole‐cell biosensor. Briefly, cells were incubated with the sample in solution for 2 h at 30 °C in 96‐well microtiter plate format, then 50 μL of 1 mM D‐luciferin were automatically injected and luminescence measurements (1 s integration) with 530–570 nm and 610–650 nm band pass filters were performed with Varioskan Flash reader (Thermo Fisher Scientific). Total androgenic activity was monitored prior to (day 0), 2, 4 and 15 days after injection and expressed as testosterone equivalents. AR activity increased 4‐5 fold two and four days after testosterone intake (p <0.0001), was back to basal activity on day 15 and, differently from conventional GC‐MS tests, was independent on the genotype. Endogenous testosterone metabolites (e.g., androsterone, etiocholanolone, testosterone‐glucuronide, 5α‐3‐α androstanediol) were also evaluated to quantify their contribution to total AR activity. As proof‐of‐concept experiment, different anabolic steroids and clinical samples were analyzed using a previously described portable device,4 relying on a disposable microwell cartridge placed in contact with a compact CCD sensor through a fiber optic taper. The androgen assay showed more precise (intra‐ and inter‐assay CV% 8 and 12%, respectively) and faster (total analysis time: 2 hrs) when compared to previously published cell‐based assays, thus showing suitable for in‐competition tests. References Michelini E, Magliulo M, Leskinen P, Virta M, Karp M, Roda A. Recombinant cell‐based bioluminescence assay for androgen bioactivity determination in clinical samples. Clin Chem. 2005;51(10):1995–8. Michelini E, Roda A. Staying alive: new perspectives on cell immobilization for biosensing purposes. Anal Bioanal Chem. In press DOI: 10.1007/s00216‐011‐5364‐x Schulze JJ, Lundmark J, Garle M, Skilving I, Ekström L, Rane A. Doping test results dependent on genotype of uridine diphospho‐glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab 2008;93, 2500–6. Roda A, Cevenini L, Michelini E, Branchini BR. A portable bioluminescence engineered cell‐based biosensor for on‐site applications. Biosens Bioelectron 2011;26:3647–53. Parvovirus B19 DNA detection in serum samples employing a microfluidic device based on chemiluminescence contact imaging M Mirasolia,b, LS Dolcia, F Bonvicinic, A Buraginaa, F Di Furioc, M Zangheria, M Guardiglia,b, G Gallinellac and A Rodaa,b* aDepartment of Pharmaceutical Sciences, University of Bologna, Bologna, Italy bDepartment of Haematology and Oncological Sciences “L. e A. Seragnoli”, Microbiology Section, University of Bologna, Bologna, Italy E‐mail: aldo.roda@unibo.it Parvovirus B19 (B19V) is responsible for a wide spectrum of pathologies and, most remarkably, it can lead to severe consequences such as fetal death if infection is contracted in pregnancy. A rapid and early diagnosis of B19V infection is thus needed to avoid potential fetal risks. A portable microfluidic device based on chemiluminescence lensless “contact imaging” detection (1,2) was employed for detecting amplified B19V DNA in serum samples and results were compared with those obtained employing a reference PCR‐ELISA method (3). The device is composed of an ultrasensitive CCD camera placed in contact, through a fiber optic taper, with a transparent microfluidics‐based reaction chip (three‐channel configuration, channel size 0.1x1x35 mm), where the capture DNA probe was covalently immobilized. After PCR amplification and biotin labelling, the DNA target sequence was denatured and added in the reaction chip, where it was captured by the immobilized probe. Hybrids were detected employing an avidin‐horseradish peroxidase (HRP) conjugate, which was revealed upon addition of a chemiluminescence substrate for HRP. Reagents flow through the channels was driven by capillary force, without use of pumps. Calibration curves, produced employing the product of amplification at different concentrations (ranging from 0.08 to 50 pmol/mL), yielded a limit of detection (calculated as the blank signal plus three times its standard deviation) of 400 fmol/mL. The method cut‐off on clinical serum samples, determined by analyzing International Standard for B19 DNA from the National Institute of Biological Standards and Control (Code 99/800), was 103 IU/mL, in accordance with standard laboratory techniques. Fifty clinical serum samples (37 positive and 13 negatives sample, as assessed with the reference PCR‐ELISA method) were subjected to the analysis and scored as negative or positive, based on the established cut‐off value. A 91.9% sensitivity and 92.3% specificity were obtained, with a positive predictive value of 97.1% and a negative predictive value of 80.0%. The device thus proved adequate for a screening method, providing reliable results with an overall assay time (after PCR amplification) of 15 min against 80 min required for PCR‐ELISA. Miniaturization of the PCR unit or isothermal DNA amplification are currently being explored to include the PCR step in the portable device. References Roda A, Mirasoli M, Dolci LS, Buragina A, Bonvicini F, Simoni P, Guardigli M. Portable device based on chemiluminescence lensless imaging for personalized diagnostics through multiplex bioanalysis, Analytical Chemistry 2011;83:3178–85. Roda A, Cevenini L, Michelini E, Branchini BR. A portable bioluminescence engineered cell‐based biosensor for on‐site applications, Biosensors and Bioelectronics, 2011;26:3647–53. Bonvicini F, Gallinella G, Cricca M, Venturoli S, Musiani M, Zerbini M. A new primer set improves the efficiency of competitive PCR‐ELISA for the detection of B19 DNA, Journal of Clinical Virology 2004;30:134–6. The role of non‐conservative Cys 62, 86, 146 and 164 residues in the functioning of Luciola mingrelica firefly luciferase YA Modestova, GY Lomakina and NN Ugarova Dept. of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia E‐mail: jmodestova@yahoo.com Though the role of the conservative Cys residues in the molecule of L. mingrelica firefly luciferase was discussed (1), the role of non‐conservative Cys residues remains unclear. Earlier we reported (2,3) that L. mingrelica firefly luciferase (WT) and its single mutants C62S, C146S and C164S possess similar specific activity, catalytic properties (Km, Vm) and spectral characteristics. Meanwhile, these mutations (especially C146S and C164S) were shown to increase the enzyme stability due to the higher effect of the reactivation process at the second stage of thermal inactivation. Using the firefly luciferase L. mingrelica carrying C‐terminal His6‐tag (WT‐His6) (4) the following mutant forms were obtained: C62S, C62V, C86S, C146S, C164S (single mutants), C62/146S, C62/164S, C86/146S, C146/164S (double mutants) and C62/146/164S (triple mutant) (Fig. 1). Comparison of the properties of mutant forms obtained on the basis of WT and WT‐His6‐luciferases revealed that the introduction of His6‐tag affected the kinetics of thermal inactivation, eliminating the first (fast) step and thus leading to the one‐step process of inactivation and the increase of thermal stability. No concentration dependence of stability was observed for any forms of His6‐luciferase. Figure 1. Spatial structure of L. mingrelica firefly luciferase. All mutants of WT‐His6 luciferase were studied in details (Table 1). The single mutations of C62S,V, C146S or C164S had no effect on the expression level, specific activity and kinetic properties, whereas the C86S substitution resulted in a drastic decrease of the expression level, specific activity and in an increase of the KmATP and KmLH2 values. The C146S mutation increased the enzyme stability at 37 °C and 42 °C, while the C164S mutation had no effect on the enzyme stability. The C62S,V mutations resulted in a slight destabilization of the luciferase both at 37 °C and 42 °C. Table 1. Properties of the L. mingrelica firefly luciferase with C‐terminal His6‐tag and its mutant forms with single, double and triple mutations of non‐conservative Cys residues Enzyme Specific activity, 1010 RLU/mg Expression level, % KmATP,μM KmLH2,μM kin, min ± 1 37° 42° WT‐His6 5.1 ± 0.4 100 310 ± 30 61 ± 5 0.022 ± 0.004 0.074 ± 0.006 C62S 4.9 ± 0.3 96 ± 5 330 ± 10 75 ± 6 0.024 ± 0.004 0.135 ± 0.004 C62V 4.5 ± 0.4 98 ± 7 350 ± 20 68 ± 5 0.036 ± 0.004 0.127 ± 0.004 C86S 1.5 ± 0.2 62 ± 6 500 ± 40 103 ± 7 0.040 ± 0.002 0.160 ± 0.006 C146S 5.8 ± 0.5 103 ± 9 290 ± 20 49 ± 4 0.011 ± 0.002 0.058 ± 0.003 C164S 5.2 ± 0.4 100 ± 7 320 ± 30 58 ± 4 0.018 ± 0.003 0.108 ± 0.005 C62/146S 4.8 ± 0.5 80 ± 6 310 ± 10 55 ± 6 0.018 ± 0.003 0.108 ± 0.005 C62/164S 3.6 ± 0.3 26 ± 5 420 ± 30 72 ± 3 0.052 ± 0.003 0.153 ± 0.005 C86/146S 1.8 ± 0.2 58 ± 5 500 ± 400 113 ± 5 0.047 ± 0.004 0.120 ± 0.006 C146/164S 4.4 ± 0.5 92 ± 8 350 ± 30 63 ± 5 0.023 ± 0.006 0.086 ± 0.005 C62/146/164S 3.2 ± 0.3 39 ± 5 400 ± 30 79 ± 3 0.055 ± 0.005 0.142 ± 0.006 The effect of double substitutions (C62/146S, C86/146S and C146/164S) on the enzyme properties was additive. The comparison of the properties of C62/164S and C62/146/164S with those of the respective single mutants revealed that the combination of the C62S/ Cys164S substitutions led to the drastic decrease of luciferase expression level, its specific activity and stability at elevated temperatures. The mechanism of these mutations influence on properties and structure of the enzyme is discussed. References Dement'eva EI, Zheleznova EE, Kutuzova GD, Lundovskikh IA, Ugarova N.N. (Physicochemical properties of recombinant luciferase from the firefly Luciola mingrelica and its mutant forms). Biochemistry (Moscow) 1996;61(1):152–9. Lomakina GY, Modestova YA, Ugarova NN. Enhancement of thermostability of the Luciola mingrelica firefly luciferase by site‐directed mutagenesis of nonconservative cysteine residues Cys62 and Cys146. Moscow University Chemistry Bulletin. 2008;63(2):63–6. Modestova YA, Lomakina GY, Ugarova NN. Temperature dependence of thermal inactivation of L. mingrelica firefly luciferase and its mutants with C62S, C146S and C164S single point mutations. Luminescence 2010;25:184–5. Koksharov MI, Ugarova NN. Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelica firefly luciferase. Photochem Photobiol Sci. 2011;10(6):931–8. Bio‐imaging of surgical stress: Dynamic analysis of liver oxidative stress and damage Naoki Moritaa, Sanae Hagab,c, Takeaki Ozawad, S. James Remingtone and Michitaka Ozakib aBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) bHokkaido University School of Medicine, Department of Molecular Surgery cThe Japan Society for the Promotion of Science (JSPS) dDepartment of Chemistry, School of Science, The University of Tokyo eInstitute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon Real‐time monitoring of cellular conditions leads to better understandings of various physio‐pathological phenomena, and will provide options in clinical diagnoses and therapies. We developed redox sensitive GFP (roGFP) and luciferase‐based caspase‐3 optical probes for in vivo imaging, and tried to visualize the dynamic changes of oxidative stress (OS) and the following damage in mouse liver ischemia/reperfusion (I/R) and partial hepatectomy (PH) models. Materials and methods A newly developed roGFP is a mutant GFP replaced with C48S, S65T, S147C, and Q204C which renders the redox sensitive property to GFP. The ratios of fluorescence from excitation at 400 and 480 nm changed in response to chemically induced OS (1). Also, we developed a novel probe (pcFluc‐DEVD) reflecting caspase‐3 activity. N‐/C‐terminal ends of firefly luciferase (Fluc) were connected to the substrate sequence (DEVD) for caspase‐3 (inactive). Once caspase‐3 is activated in cells (DEVD is cleaved), Fluc changes into an active form, restoring luminescence activity (2) (Fig. 1). Figure 1. Principle for monitoring the caspase‐3 activity using cyclic firefly luciferase. By transfecting adenovirus vectors coding for roGFP (AdroGFP) or pcFluc‐DEVD (AdpcFluc), we investigated whether these probes will monitor redox states and apoptosis in live cells and liver during hypoxia/reoxygenation (H/R), I/R and PH in mice (3,4,5). Results roGFP visualized H/R‐induced dynamic changes of cellular redox states. Cellular redox was slightly reduced during hypoxia, but was rapidly and transiently oxidized during post‐reoxygenation. roGFP well illustrated the anti‐oxidative effects of N‐acetyl cysteine, catalase, and Ref‐1 on H/R‐induced cellular OS. Similarly, pcFluc‐DEVD probe reflected cellular caspase‐3 activity induced by various pro‐apoptotic stimuli dose‐dependently (FasL, staurosporine and H/R). These probes also illustrated the redox changes by the repeated stimuli, indicating that these probes functioned reversibly. In mouse liver I/R experiment, adenovirally transfected roGFP showed two peaks of OS in the post‐ischemic liver. The early OS peak, originating from liver cells, was observed within 60 min, and increased its intensity in proportion to the ischemic time and the following liver injury. The second and larger peak of OS, which originates from infiltrating neutrophils, was observed 24 hr post‐ischemia or later. pcFluc‐DEVD probe indicated on‐going processes of liver damage quantitatively by visualizing the dynamic changes of caspase‐3 activities in the post‐ischemic liver (Fig. 2). These probes together revealed time‐/strength‐relationships of OS and damages in I/R model. Figure 2. Monitoring of caspase‐3 activity in the post‐ischemic liver in a mouse (left & middle liver ischemia and reperfusion). Summary The roGFP redox‐ and pcFluc‐DEVD caspase‐probes successfully illustrated OS and the following damage in vitro and in vivo. By visualizing the organ conditions, these may provide many options for diagnoses and treatments in the future. References Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 2004;279:22284–93. Kanno A, Umezawa Y, Ozawa T. Detection of apoptosis using cyclic luciferase in living mammals. Methods Mol Biol 2009;574:105–14. Haga S, Terui K, Fukai M, Oikawa Y, Irani K, Furukawa H, Todo S, Ozaki M. Preventing hypoxia/reoxygenation damage to hepatocytes by p66shc ablation: Up‐regulation of anti‐oxidant and anti‐apoptotic proteins. J Hepatol 2008;48:422–32. Haga S, Remington SJ, Morita N, Terui K, Ozaki M. Hepatic ischemia induced immediate oxidative stress after reperfusion and determined the severity of the reperfusion‐induced damage. Antioxid Redox Signal 2009;11:2563–72. Haga S, Morita N, Irani K, Fujiyoshi M, Ogino T, Ozaki M. p66Shc has a pivotal role in impaired liver regeneration in aged mice by a redox‐dependent mechanism. Lab Invest (2010);90:1718–26. ANTIRADICAL CAPACITY EVALUATION OF EXTRACTS AND INFUSIONS FROM Baccharis regnelli USING A CHEMILUMINESCENCE ASSAY PROCEDURE Flávia U. de Matosa, Sandro de Oliveirab, Marcelo J. P. Ferreiraa, Oriana A. Fáveroa, Norberto P. Lopesc, Wilhelm J. Baaderb and Paulete Romoffa* aCentro de Ciências e Humanidades e Centro de Ciências e Biológicas e da Saúde, Universidade Presbiteriana Mackenzie, CEP 01302‐907, São Paulo ‐ SP, Brazil bInstituto de Química, Universidade de São Paulo, CEP 05508‐900, São Paulo ‐ SP, Brazil cFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, CEP 14040‐903 Ribeirão Preto ‐ SP, Brazil Baccharis genus is widespread in tropical areas of South America, and comprises about 500 species, of which 120 occur in Brazil, and many of them are used in folk medicine to treat a wide variety of diseases. In this sense we propose a screening test for antiradical activity of extracts, separation phases and chromatographic fractions, using the antiradical capacity as a preliminary parameter for potential biological activity. In the present work we report the antiradical activity of the hydroalcoholic extracts and infusions obtained from the aerial parts of Baccharis regnelli collected in Campos do Jordão, São Paulo. The antiradical capacity was determined using the luminol chemiluminescence antiradical assay developed by our research group.(1) The resulting antiradical capacity of the complex mixtures were expressed in relation to trolox, using percentage trolox values (% trolox), which are directly proportional to the antiradical activity of the sample, contrarily to the TRAP values which are inversely proportional to the activity. Therefore the % trolox values suggested here appear to be more convenient than TRAP values to compare antiradical activity of complex mixtures. The antiradical capacity guided study of Baccharis regnellii aerial parts lead to the identification of chlorogenic acid derivatives in the hydroalcoholic extract. Financial support: Fundo Mackenzie de Pesquisa, FAPESP, CNPq. ___________________________________________________ 1 Bastos EL, Romoff P, Eckert CR, Baader WJ. J. Agric. Food Chem. 2003;51:7481. A novel eukaryotic‐cell based bioluminescence assay for detection of oxidative‐stress inducing compounds Paria Motahari, Mehrdad Behmanesh and Majid Sadeghizadeh* Department of Genetics, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran *Corresponding Author's email: sadeghma@modares.ac.ir Introduction The modern life of human is accompanied by encounter with various environmental stresses which lead to the accumulation of free radicals in the body. These free radicals give rise to damages to cellular biopolymers including nucleic acids, proteins, carbohydrates and fatty acids. Hence, the goal of our work was to develop a biosensor with high sensitivity and specificity able to detect a wide range of compounds with oxidative capacity in biological samples. Materials and methods Two single‐stranded oligonucleotides containing the ARE core promoter, after annealing, were cloned into PGL4.26 vector, which comprise the luc2 gene encoding luciferase as reporter. Following amplification of the vector harboring the ARE sequence in E. coli, it was transfected into HUH7 cells (human hepatoma cells) grown in DMEM medium. After 24 hours, the cells were treated with two chemicals (hydroquinon and benzoquinone each with 10 μM concentration). Next, luciferase assay was performed. Results The data obtained here exhibited that the ARE sequence is active in the presence of oxidative stress‐inducing compounds hydroquinone and benzoquinone. Also, it was revealed that he HUH7 cells treated with hydroquinone and benzoquinone possess a higher level of luciferase expression, up to 10 and 6 times more, respectively, in comparison with the control cells which were not exposed to oxidative‐stress inducers. Discussion According to the data obtained in the first phase of our study presented here we were able to construct a transient eukaryotic‐cell based biosensor which has the capability to detect hydroquinone and benzoquinone compounds in the medium. The next phase of our study has been focused on the construction of a permanent biosensor with the ability of detecting a wide variety of oxidative‐stress inducing chemicals. The results will be presented. Electrochemiluminescent Determination of Free Unconjugated Bilirubin in Aquatic Solution Kateryna Muzyka, Olena Bilash, Yuriy Zholudov, Anatoly Kukoba and Mykola Rozhitskii Laboratory of Analytical Optochemotronics, Kharkiv National University of Radio Electronics,14 Lenin Ave, 61166, Kharkiv, Ukraine; E‐mail: rzh@kture.kharkov.ua Bilirubin (formerly referred to as hematoidin) is the yellow breakdown product of normal heme catabolism. It is generally accepted that unconjugated bilirubin (UCB) bound to albumin or other plasma (lipo)proteins is not toxic. UCB is a subject of interest in neurotoxicology and other biomedical fields. Unbound free UCB is toxic and may bound to brain cells, when the concentration is higher than its aqueous solubility (70 nM) under physiological condition (pH 7.4). However, free UCB concentrations of 40 nM (i.e. below aqueous solubility) also showed toxicity to cultured astrocytes (1). Since damage to neurons and astrocytes by bilirubin can occur at free UCB concentrations near or smaller then 70 nM concentration limit it is essential to develop efficient and cheap method for nanomolar concentration of free UCB determination in water solution. Such definition is not accessible by widespred techniques as spectroscopic and electrochemical one. The main purpose of the present work is the electrogenerated chemiluminescence (ECL) determination of free UCB in aqueous solution in view of different applications (clinical, biomedical etc.). ECL is the process, where reactant species generated at electrodes of ECL cell undergo high‐energy electron‐transfer reactions to form product excited states that emit light. ECL assay is powerful and a very widely used method of analysis of different objects (2). However, there is no information about ECL determination of UCB. Therefore, in this work we study for the first time the possibility of ECL determination of free UCB. The coreactant nature of the involvement of bilirubin as a coreactant in the homogeneous ECL reactions with electrogenerated ion radicals of trisbipyridine ruthenium was shown. Electrochemical behavior and modes of electrochemical excitation of ECL in model water solutions with bilirubin using glassy carbone electrode with a diamond‐like, as well as the influence of pH and UCB concentration on the analytical signal were investigated. The calibration curve for the ECL determination was obtained that showed the limit of UCB detection in water solutions is about 1 nM. So the main conclusion of our investigation is proved experimentally the possibility of nanomolar UCB concentration ECL determination. This work was supported by Science and Technology Centre in Ukraine (STCU) Project #5067 (Project Manager – Prof. M. Rozhitskii). References Ostrow JD, et al. Molecular basis of bilirubin‐induced neurotoxicity. Trends Mol Med 2004;10:65. Rozhitskii NN. Zhurn. Anal. Khim. 1992;47:1765. Light emission in firefly: a theoretical study Isabelle Navizeta, Ya‐Jun Liub, Nicolas Ferréc, Shu‐Feng Chenb,c, Hong‐Yan Xiaod and Roland Lindhe aSchool of Chemistry, University of the Witwatersrand, PO Wits Johannesburg, 2050, SOUTH AFRICA E‐mail: isabelle.navizet@wits.ac.za bKey Laboratory of Theoretical and Computational Photochemistry, College of Chemistry, Beijing Normal University, Beijing 100875, CHINA cLaboratoire Chimie Provence, Universités d'Aix‐Marseille I, II et III‐CNRS UMR 6264, Case 521, 13397 Marseille Cedex 20, FRANCE. dTechnical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, CHINA eDept. of Chemistry ‐ Ångström, the Theoretical Chemistry Programme, Uppsala University, P.O. Box 518, S‐75120 Uppsala, SWEDEN The emitting light in fireflies arises from the electronic relaxation of oxyluciferin, an organic compound resulting from the oxidation of the luciferin substrate inside an enzyme called luciferase. The color of the emitted light can be modulated by mutation of the luciferase. We used quantum mechanical/molecular mechanical (QM/MM) methods, implemented in MOLCAS and TINKER, to clarify the relationship between the structure of the system and the color of the emitted light. The use of the complete active space SCF (CASSCF) and the multi‐configurational reference second‐order perturbation theory (CASPT2) is required to study of the electronic system and the use of molecular mechanics (MM) to take into account the surrounding molecules. Our systematic theoretical investigation of all the possible light emitter structures of firefly shows that the phenolate‐keto form of oxyluciferin is responsible for the light emission (1). Our theoretical results (2) on the oxyluciferin‐luciferase complex shows in agreement with recent experimental observations that the wavelength of the emitted light depends on the polarity of the microenvironment at the phenol/phenolate terminal of the benzothiazole fragment of oxyluciferin. Color modulation can be obtained by mutation of the luciferase or the luciferin on the benzothiazole fragment. References Chen SF, Liu YJ, Navizet I, Ferré N, Fang WH, Lindh R. J. Chem. Theory Comput. 2011;7(3):798–803. Navizet I, Liu Y‐J, Ferré N, Xiao H‐Y, Fang W‐H, Lindh R. J. Am. Chem. Soc. 2010;132(2):706–12. The Chemistry of Bioluminescence: An Analysis of Chemical Functionalities Isabelle Navizeta, Ya‐Jun Liub, Nicolas Ferréc, Daniel Roca‐Sanjuán, Mickaël Delceyd and Roland Lindhd aUniversity of the Witwatersrand, South Africa bBeijing Normal University, China cUniversités d'Aix‐Marseille, France dUppsala University, Sweden Firefly luciferase is one of the most studied bioluminescent system. It has been extensively studied both theoretically and experimentally. Based on these studies we will herein give a review on the current understanding of the bioluminescent process from a chemical functionality perspective. This presentation will emphasize three key components: the chemiluminophore, the electron‐donating fragment and how these are affected by the substrate‐enzyme interaction. The understanding is based on details of how the peroxide ‐O‐O‐ bond supports the production of electronically excited products and how the Charge Transfer Induced Luminescent, CTIL, mechanism, with the aid of an electron‐donating group, lowers the activation barrier, to support a reaction in living organisms. For the substrate‐enzyme complex it is demonstrated that the enzyme can affect the hydrogen‐bonding around the CTIL controlling group resulting in a mechanism for color modulation. Finally, in the light of the purpose of the fragments of the luciferin‐luciferase complex to provide key chemical functionalities we will analyze other luciferin‐luciferase systems with respect to similarities and differences. NanoLight Technology‐based Probe, a Useful Tool for Detection of Apoptotic Cells Mahbobeh Nazaria , Rahman Emazadehb*, Saman Hosseinkahnic, Luca Ceveninid, Elisa Michelinid and Aldo Rodad aNanobiotechnology Research center (NBRC), Avicenna Research Institute (ARI), Shahid Beheshti University, Tehran, Iran bDepartment of Biology, Faculty of Science, University of Isfahan, Tehran‐Iran cDepartment of Biochemistry, Faculty of biological sciences, Tarbiat Modares University, Tehran, Iran dAnalytical and Bioanalytical Chemistry Laboratory, Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy E‐mail: Sci_rahman@yahoo.com Apoptosis is the process of programmed cell death that can occur in multicellular organisms and it may be employed as a target in the clinic to treat diseases (1). In fact, many anti‐cancer agents act by the induction of the apoptosis in sensitive tumour cells. During the early stages of apoptosis, the asymmetric distribution of plasma membrane phospholipids is lost, which results in exposure of phosphatidylserine on the extracellular leaflet of cell membrane. This phenomenon can be detected by annexin‐based probes. (2 and 3) In this study, a new probe for detection of apoptosis based on the annexinV in fusion with a reporter protein from nano‐light technology (Nanolight Corporation) has been designed, tested and developed. Figure 1. Apoptosis assay. The signal to noise ratio was significantly increased up to 5‐fold within 16 hours after induction of apoptosis by actinomycine D. The probe was constructed using protein fusion technology, expressed and purified using Ni Sepharose. Moreover, CHO cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) at 37 °C in a humidified atmosphere containing 5% CO2. Apoptosis was induced by an anti‐cancer drug (actinomycin D). Finally, apoptotic cells were treated by the nanoprobe and apoptotic signals were collected and analyzed. The result was shown that the signal to noise ratio increased up to 5‐folds after induction of apoptosis (Figure 1). In conclusion, in this research a new probe based on the nano‐light technology from the Nanolight Corporation for detection of apoptosis is produced and assayed for functional activity. This is a fast, sensitive and non‐invasive procedure for apoptosis assay and besides the commercial products; it might be used as an alternative probe for screening of anti‐cancer agents. References Susin SA, Lorenzo HK, Zamzami N. Molecular characterization of mitochondrial apoptosis‐inducing factor" Nature 1999;397:441–6. Logue SE, Elgendy M, Martin SJ. Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Nature Protocols 2009;4:1383–95. Strebel A, Harr T, Bachmann F, Wernli M, Erb P. Green fluorescent protein as a novel tool to measure apoptosis and necrosis. Cytometry 2001;43:126–33. Effect of Ethylenediamin bispyridine copper(II) perchlorate as an enhancer of luminol chemiluminescence O Nazaria*, A Ehteshama, MJ Chaichia, H Golchoubiana and J Mehrzadb aFaculty of Chemistry, Mazandaran University, Babolsar, Iran bFaculty of Veterinary Medicine, Ferdowsi University, Mashhad, Iran *Corresponding author e‐mail: omnazari@yahoo.com Some transition metal complexes are introduced as enhancer in the luminol chemiluminescence (CL) system (1–3). In this work, the catalytic effect of the synthesized copper (II) complex type (Cu(en)Py2)(ClO4)2 with the structure confirmed by X‐ray crystallography, on luminal was investigated (Figure 1). The X‐ray crystal analysis of the complex demonstrated that the copper(II) ion is in square planar environments through coordination by two nitrogen atoms of the ethylenediamine and two nitrogen atoms of two pyridine molecules and the ClO4− ions are bound weakly above and below of the molecular plane(4). It can catalyze and enhance luminol CL. The light intensity of luminol‐H2O2 system is increased with increasing of the complex concentration. The effect of hydrogen peroxide, luminol and the complex concentration on CL are investigated. Moreover we compared the enhancing effect of the complex with some other luminol CL enhancers such as copper (II), ferrous and cobalt ions. The complex can be used for determination of hydrogen peroxide in low concentration. Figure 1. Chemical structure of the complex. References Parejo I, Petrakis C, Kefalas P. A transition metal enhanced luminol chemiluminescence in the presence of a chelator. Journal of Pharmacological and Toxicological Methods 2000;43:183–90. Hanaoka S, Lin JM, Yamada M. Chemiluminescent flow sensor for H2O2 based on the decomposition of H2O2 catalyzed by cobalt(II) ethanolamine complex immobilized on resin. Anal. Chim. Acta 2001;426:57–64. Tahereh Khajvand, Mohammad Javad Chaichi, OmLeila Nazari and Hamid Golchoubian, Application of Box–Behnken design in the optimization of catalytic behavior of a new mixed chelate of copper (II) complex in chemiluminescence reaction of luminal. Journal of Luminescence 2011;131:838–42. Hamid Golchoubian, Omeleila Nazari and Benson Kariuki, Synthesis, Structure and Solvatochromism Studies on Copper(II) Complexes Containing Ethylenediamine, Pyridine and Imidazol Ligands, Journal of the Chinese Chemical Society 2011;58:60–8. Quantum yield and kinetics of the bioluminescence reaction using various beetle luciferases Kazuki Niwa, Yoshiro Ichino, Mika Maenaka, Takaya Kubo, Yoshihiro Hiraishi, Dai‐ichiro Kato and Yoshihiro Ohmiya National Metrology Institute of Japan / AIST, 1‐1‐1 Umezono, Tsukuba, 305‐8563 Japan E‐mail: niwa‐k@aist.go.jp Firefly bioluminescence reaction is well‐known for its extremely high quantum yield (QY) value(1). QY, as well as other kinetic parameters, is a quite important factor to determine the brightness of the luminescence reaction. These factors for beetle luciferases were not uniformly explored, although the wide variation of the catalytic character of the enzyme. Especially, spectra of the luminescence reaction vary for individual luciferases and/or reaction conditions. For example, the railroad worm, Phrixothrix hirtus, has a unique red bioluminescence color. For pH‐sensitive luciferase, such as from Photinus pyralis, Pylocoeria miyako, and so on, emission spectrum of the reaction solution is influenced by pH condition of the solution. Mutations in luciferases can also change the color of the emitted luminescence. Here, we explored QY value and kinetic parameters using these various luciferases. Wild‐type luciferases were prepared as His‐Tag recombinant protein. Mutant luciferases of P. miyako were also prepared to compare with their wild‐type enzymes. QY was measured using a luminometer whose absolute responsivity (sensitivity) to the total number of photons emitted from the luminescence reaction solution in a test tube was calibrated in accordance with a method we had reported (2). The Km for the substrate D‐luciferin and VMax were calculated from Lineweaver–Burk plots, followed by the calculation of kcat which is the turnover number of the reaction for the substrate D‐luciferin by a single luciferase molecule per second. QY for native P. pyralis luciferase (0.45) was consistent with previous reports (1, 2) within the range of uncertainty. Pyrearinus termitilluminans luciferase, whose luminescence peak wavelength was the shortest of the luciferases examined, had the highest QY (0.61), whereas P. hirtus red‐colored luciferase had the lowest QY value (0.15). QY values for mutant luciferases were almost identical if the peak wavelength was consistent with that for wild‐type. In contrast, QY values for most of the red‐shifted mutant decreased as the peak wavelength shifted longer (Figure 1). These results suggest that the QY and the color of bioluminescence reaction are strongly related to each other. Figure 1. Quantum yield and luminescence peak energy for beetle luciferases. Despite the differences of QY value, its variation is only within the same order. This fact was also similar to Km and kcat values for wild‐type luciferases. These results indicate that the enormously wide variation in the light intensity emitted from the bioluminescence reaction samples using wild‐type luciferases can only be explained by variation in the concentration and/or the stability of the enzyme. In contrast, kcat for mutant luciferases decreased significantly, which would result in reducing the light intensity. We conclude that the effective means to obtain a stronger luminescence signal using a mutant luciferase would be to increase the concentration and stability of the enzyme but not to decrease kcat (3). References Ando Y, Niwa K, Yamada N, Enomoto T, Irie T, Kubota H, Ohmiya Y, Akiyama H. Firefly bioluminescence quantum yield and colour change by pH‐sensitive green emission. Nat. Photonics 2008;2:44–7. Niwa K, Ichino Y, Ohmiya Y. Quantum yield measurements of firefly bioluminescence reactions using a commercial luminometer. Chem. Lett. 2010;39:291–3. Niwa K, Ichino Y, et al. Quantum yields and kinetics of the firefly bioluminescence reaction of beetle luciferases. Photochem. Photobiol. 2010;86:1046–9. Modulation of luminescence intensity of whole non‐diluted human blood by hydrated fullerenes in ultra‐low doses Kirill N. Novikova*, Olga I. Yablonskayaa, Nadezhda G. Berdnikovab and Alexey K. Novikovb, Ekaterina V. Bouravlevaa, Vladimir L. Voeikova aLomonosov Moscow State University, Faculty of Biology; bSechenov First Moscow State Medical University, Moscow, Russia *E‐mail: kirniknov@yandex.ru Previously we found that after addition of luminescent probes for reactive oxygen species (ROS) – luminol (LM) or lucigenin (LC) – long‐lasting weak photon emission from non‐diluted whole blood may be observed (1). Here we studied effects of hydrated fullerenes C60 (HyFn) in concentrations in the range of 10−6–10−19 M, upon LC‐amplified chemiluminescence (CL) of whole human blood. We found that intensity of LC‐dependent CL from fresh healthy donors' blood increases 3‐5‐fold after addition of HyFn in concentrations 10−6 and 10−17–10−19 M while in concentrations 10−7–10−15 M HyFn practically did not affect intensity of LC‐dependent CL (Figure 1). Figure 1. Effect of HyFn in high dilutions upon intensity of LC‐dependent CL in blood of healthy donors. On the other hand 24 hours after LC and HyFn have been added to blood CL intensity in it was much higher than in control blood. Thus HyFn stabilizes its non‐equilibrium state of blood. Intensity of LM‐zymosan‐ and LC‐dependent blood CL of chronic obstructive pulmonary disease (COPD) patients was much higher before the Hypoxen® treatment and significantly decreased after successful therapy. Addition of HyFn (10−6 и 10−19 M) to blood of patients strongly attenuated blood CL. After treatment HyFn diminished photon emission to much lower degree or did not affect it (Figure 2). Figure 2. Effects of HyFnC60 on Luminol and luminol+‐zymosan‐ (a) and lucigenin‐ (b) dependent CL of nondiluted blood of COPD patient T. at HyFnC60 action. 1‐ before Hypoxen® treatment, 15.10.09, no HyFnC60; 2 – 1 + HyFnC60 (10‐19 M); 3 ‐ after Hypoxen® treatment, 22.12.09, no HyFnC60; 4 ‐ 3 + HyFnC60 (10‐6 M); 5 ‐ 3 + HyFnC60 (10‐19 M). Modulation of HyFn in ultra‐low concentrations of free radical processes going on in blood may be due to modification of aqueous matrix of blood by HyFn, because fullerenes C60 being chemically inert nanoparticles are covered in HyFn preparations with multilayer water “shells” (2). Such an organized aqueous phase may serve as a donor of electrons and thus a modifier of redox properties of blood (3). Opposite action of HyFn on such processes in healthy donors' and patients' blood may be used for diagnostic purposes. Besides it indicates that HyFn may possess normalizing action on free radical processes that are the necessary part of metabolic activity of all living organisms. References Voeikov VL, Asfaramov R, Bouravleva EV, Novikov CN, Vilenskaya ND. Biophoton research in blood reveals its holistic properties. Indian J. Exp. Biol. 2003;43:473–82. Andrievsky GV, Bruskov VI, Tykhomyrov AA, Gudkov SV. Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in vivo. Free Rad. Biol. Med. 2009;47:786–93. Voeikov VL, Del Giudice E. Water respiration – the basis of the living state. WATER: A multidisciplinary Res. J., 2009, 1, 52–75. http://waterjournal.org/content/view/45/64/. Spectrum of the chemiluminescence emission from the heme‐catalyzed imidazole chemiluminescence Osamu Nozakia, Hidehiro Kubotab, Motohiro Shizumac, Motonori Munesued and Tadasu Ikedae aDepartment of Clinical Laboratory Medicine, Kinki University School of Medicine. Osaka‐Sayama 589‐8511, Japan bScientific division, ATTO Co., Tokyo 111‐0041, Japan cTechnochemistry Department, Osaka municipal technical research institute, Osaka 536‐8553, Japan dTechnical Department, ChemcoPlus Scientific Co.LTD, Higashi‐Osaka 577‐0065, Japan eSchool of Medicine, Tottori University, Yonago 683‐8503, Japan. Imidazole chemiluminescence (CL) emits light with imidazole analogues, peroxide and oxidative catalyst. Light spectrum shows the characteristics of chemiluminescence. There were no reports of light spectrum of imidazole CL. Therefore, the spectrum of chemiluminescence was investigated with different kind of catalysts (horseradish peroxidase, cytochrome C, hemoglobin, hemin, and ferricyanide) in this study. Spectra of imidazole CL with heme (HRP, cytochrome C, hemoglobin, and hemin) showed two peaks (max. = 510 and 620 nm) ranging from 420 to 700 nm (Figure 1). The two peaks meant existence of two kinds of light sources. We considered the sources of the two lights as follows. One was due to imidazole hydroperoxide. Because when no imidazole and hydrogen peroxide existed, no light emitted. The two peak heights changed in correlation to amount of hydrogen peroxide. Amount of imidazole hydroperoxide that was a product of imidazole and hydrogen peroxide, decreased in time course of imidazole CL reaction. The other light source was due to energy transfer from the imidazole reaction to heme (speculation). Because, imidazole CL catalyzed with ferricyanide showed chemiluminescence spectrum of only one peak (max. = 510 nm) (Figure 2). The absorption spectra with heme proteins (HRP, cytochrome C, and hemoglobin) showed two peaks (max. = 540 and 560 nm) at beginning of imidazole CL reaction, and the heights of the peaks diminished in course of the CL reaction. The second peak of CL spectrum (max. 620 nm) appeared as the result of energy transfer from reaction of imidazole hydroperoxide (light of max. 510 nm). In conclusion, the light spectra of heme catalyzed imidazole CL showed two peaks revealing existence of two light energy sources (imidazole hydroperoxide and heme energy transferred). Heme emitted light in combination with imidazole chemiluminescence. Figure 1. Light spectrum by HRP catalyzed imidazole CL. Figure 2. Light spectrum by ferricyanide catalyzed imidazole CL. Bioluminescence in vivo Imaging using a Cypridina Luciferase Yoshihiro Ohmiyaa and Chun Wub aBioproduction Research Institute, National Institute of AIST, Higashi 1‐1‐1, Tsukuba, Ibaraki, Japan 305‐8566 bHealth Research Institute, National Institute of AIST, Midorioka 1‐8‐31, Ikeda, Osaka, Japan 563‐8577 An increasing number of monoclonal antibodies have been used to target antigens on cancer cells for clinical diagnosis and therapy, based on the fact that some antigens expressed on cancer cells surface reflect malignant behaviors invasion, metastasis, and neo‐vascularization. Molecular imaging of antibodies in the whole body will enable us to prescribe the appropriate antibody therapy in terms of dose and the timing of administration. Bioluminescence imagings (BLI) have played an important role in molecular imaging in small animals. BLI is achieved with a luciferin‐luciferase reaction in the presence of molecular oxygen. However, most bioluminescence spectra are in the visible region, overlapping with the absorption spectrum of hemoglobin, attenuating the bioluminescence intensity in live animals. On the other hand, Cypridina luciferase (CLuc) catalyzes the oxidation of Cypridina luciferin to yield light emission peaking at 460 nm. The 62‐kDa CLuc has some unique properties as a bioluminescent enzyme. The secreted protein contains 17 disulfide bond pairs and is highly stable under physiological conditions. Its turnover rate (1,400 luciferin molecules per minute) is the highest among known luciferases. Recently we have established a method for the synthesis of the substrate, and have expressed the recombinant CLuc in yeast and applied it to ELISA (1–3). Furthermore, we conjugated a far‐red fluorescent indocyanine derivative to biotinylated CLuc via glycol‐chains and named this far‐red bioluminescent protein “FBP” (Figure 1). FBP probe, having a bimodal spectrum (λmax = 460 nm and 675 nm), are extremely stable under different conditions of pH and ion concentration. Using anti‐tumor monoclonal antibody linked to FBP via biotin‐avidin interaction, we achieved bioluminescence imaging of cancer cells in vivo as well as in vitro (4). So, this FBP offers a very useful analytical tool for the evaluations of monoclonal antibody localization in live animals. Figure 1. Utility of FBP labeling covering in vitro‐, in vivo‐ and ex vivo‐imaging. References Wu C, Kawasaki K, Ohgiya S, Ohmiya Y. Syntheses and evaluation of the bioluminescent activity of (S)‐Cypridina luciferin and its analogs. Tetrahedron Lett. 2006; 47:753–756. Wu C, Kawasaki K, Ogawa Y, Yoshida Y, Ohgiya S, Ohmiya Y. Preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay. Anal Chem 2007;79:1634–8. Wu C, Irie S, Yamamoto S, Ohmiya Y. A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase. Luminescence 2009;24:131–3. Wu C, Mino K, Akimoto H, Kawabata M, Nakamura K, Ozaki M, Ohmiya Y. In vivo far‐red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye. Proc Natl Acad Sci USA 2009;106:15599–603. On the purification of the fungal luciferin Anderson G. Oliveiraa, Rodrigo P. Carvalhob and Cassius V. Stevanib aDept. de Genética e Evolução, Universidade Federal de São Carlos, Sorocaba, SP, Brazil bDept. de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil Since the early 20th century, many researchers have attempted to determine how fungi are able to emit light. However, the uncertainty about the possible involvement of a luciferase in fungal bioluminescence has not only hindered the understanding of its biochemistry but also delayed the characterization of its constituents. Thus, despite some efforts made in past decades, the fungal luciferin has not been elucidated. The present work describes how in vitro light emission can be obtained enzymatically from hot and cold extracts, using different species of luminous fungi, in order to purify the fungal luciferin. Moreover, we report our progress in the luciferin purification using HPLC, NMR and LC/MS techniques. A single luminescent system in all fungal bioluminescent lineages Anderson G. Oliveiraa, Dennis E. Desjardinb*, Brian A. Perryc and Cassius V. Stevanid aDept. de Genética e Evolução, Universidade Federal de São Carlos, Sorocaba, SP, Brazil bDept. of Biology, San Francisco State University, San Francisco, CA, USA cDept. of Biology, University of Hawaii at Hilo, Hilo, HI, USA dDept. de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil All luminescent systems involve the catalytic oxidation of a substrate (luciferin) by a respective enzyme (luciferase). However, despite the progress achieved in the last century in understanding biological and evolutionary aspects of various bioluminescent systems, there are still some luminous organisms that remain poorly understood. Bioluminescent fungi are an example. The Fungi kingdom is represented by about 1.5 million species, but of this diversity only 71 species are luminous and they belong to four distantly related lineages: Armillaria, Lucentipes, Mycenoid and Omphalotus. The uncertainty about a luciferase presence in fungal BL hampered for decades the understanding of the biochemical pathways involved in light emission. This work aims to show that the mechanism of fungal BL, and its components (luciferin, luciferase and NAD(P)H reductase), are similar in all known strains of bioluminescent fungi suggesting a single evolutionary origin in BL fungi. To corroborate this idea, hot extracts were prepared (luciferin source) and cold extracts (luciferase source) using mycelia and fruiting bodies of BL fungi belonging to four lineages and non bioluminescent fungi as well. The study indicates all four lineages of luminescent fungi share the same type of luciferin and luciferase, that there is a single luminescent mechanism in the Fungi. On the purification of the NAD(P)H‐dependent reductase involved in fungal bioluminescence Anderson G. Oliveiraa, Rodrigo P. Carvalhob, Hans E. Waldenmaierb, Vadim R. Viviania and Cassius V. Stevanib aDept. de Genética e Evolução, Universidade Federal de São Carlos, Sorocaba, SP, Brazil bDept. de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil Oliveira and Stevani successfully confirmed the enzymatic nature of fungal bioluminescence after nearly fifty years since the initial report of Airth and Foerster. This confirmation was achieved by using the Dubois' classical luciferin‐luciferase assay that consists of mixing under controlled conditions hot (source of luciferin) and cold (source of enzymes) water extracts prepared from bioluminescent fungi in the presence of NAD(P)H. As previously evidenced by Airth and Foerster, the cold extract contains a NAD(P)H‐dependent reductase and a luciferase, whose initial purification could be accomplished by ultracentrifugation. In order to purify the reductase, a solution of the hot extract and NAD(P)H has been used to evidence its presence in the purifying fractions. In this work, we report our progress in the purification of the fungal reductase involved in fungal bioluminescence by conventional techniques as ultracentrifugation, exclusion and affinity chromatography, and native PAGE. Bioluminescence and fluorescence in scale‐worms (Polychaeta, Polynoidae) M Plyuschevaa, A Goñib, V Saprunovac and F Kondrashova aCentre de Regulació Genòmica (CRG) Barcelona, Spain bInstitut de Ciència de Materiales de Barcelona (ICMAB, CSIC), Bellaterra, Catalunya, Spain cA.N.Belozersky Institute of Physico‐Chemical Biology, Moscow State University, Moscow, Russia Fluorescence is found in an unaccountably diverse array of marine organisms. Iit was hypothesized that ocean inhabitants would be able to use fluorescence for visual communication and signaling. The spectrum of the bioluminescent emissions is often the same as that of the fluorescent ones. The currently known photoproteins are not fluorescent before reaction and their bioluminescence‐excited states are the same as their fluorescence ones. As for the scale‐worms, “bioluminescent” species, like Acholoe astericola, show a yellowish fluorescence in UV light after subsiding of bioluminescence, while the “non‐bioluminescent”, like Lepidonotus clava, show only a bluish fluorescence in the skeletal parts (Harvey, 1952). Bioluminescent properties, fluorescence distribution and in vivo spectral characteristics were analyzed among five “bioluminescent” species and two “non‐bioluminescent”. The fluorescence was measured using a high‐resolution, high‐throughput grating spectrometer equipped with a charge‐coupled device (CCD) detector in combination with a confocal microscope. Several sources of fluorescence were observed in all studied “bioluminescent” species. First source is so called by Bassot “photogenic area” (Bassot & Nicolas 1995), cells of ventral epithelium, surrounding place of attaching elytra to elytrophore (Fig. 1A). This zone visible from the ventral side of elytra and fluorescence signal is very bright. From the dorsal side all elytra of scale worms covered by tubercle. Excited with UV light tubercles are found to show bright bluish fluorescence in both “bioluminescent” and “non‐bioluminescent” species (Fig. 1B). The third source of fluorescence in “bioluminescent” species is located at the dorsal side of elytra in dorsal epithelial cells. This source can be observed after several repetitions of bioluminescent stimulations. Figure 1. Sources of fluorescence in scale‐worms. A – ventral “photogenic area” of bioluminescent Harmothoe fragilis, in the background tubercles from dorsal side. B – dorsal side of elytra, covered with tubercles, of non‐bioluminescent Lepidonotus clava. Ex 488 nm. In all studied species were observed the same emission spectrum. With UV excitation there is a narrow peak at 506–515 nm maximum and a wide shoulder around 600 nm maximum. With 514 nm excitation emission is in the orange/red part of the visible spectrum at about 600 nm. The presence of polynoidin in “non‐luminescent” species suggests that the ability to bioluminescence has been lost and that bioluminescence is a function of morphological adaptation (Plyuscheva & Martin 2009). References Bassot J‐M, Nicolas MT. Bioluminescence in scale‐worm photosomes: the photoprotein polynoidin is specific for the detection of superoxide radicals. Histochemistry and Cell Biology 1995;104(3):199–210. Harvey EN. Bioluminescence., New York: Academic Press, 1952. Plyuscheva M, Martin D. Morphology of elytra as luminescent organs in scale‐worms (Polychaeta, Polynoidae). Proceedings of the Ninth International Polychaete Conference, Zoosymposia 2009;2:379–89. Cloning, expression, and purification of the photoprotein responsible for luminescence in the deep‐sea ctenophore Bathocyroe fosteri Meghan L. Powersa and Steven Haddockb aUniversity of California at Santa Cruz, b Monterey Bay Aquarium Research Institute Calcium‐binding photoproteins have been discovered in a variety of luminous marine organisms. Light emission occurs when calcium binds to a photoprotein‐substrate‐oxygen complex where the substrate, usually coelenterazine, is oxidized to produce blue light. This group of photoproteins has been widely studied in hydrozoans which use the same general mechanism and have similar spectral properties. However, to further understand the evolution of these proteins and their potentially unique properties, more primary sequence information is needed. Recent interest in this area has led to the identification of several ctenophore photoproteins. Here we report the cloning, expression, and purification of the photoprotein responsible for luminescence in the deep‐sea ctenophore Bathocyroe fosteri. This animal was of particular interest due to its unique dual color spectrum observed in live specimens. Full‐length sequences were identified using known photoprotein sequences to BLAST Bathocyroe expressed sequence tags (ESTs) obtained from 454 transcriptome sequencing. Primary structure alignment of the Bathoocyroe photoprotein with both mnemiopsin 1 and 2, berovin, and bolinopsin showed very strong sequence similarity and conservation of Ca2+ binding sites. Preliminary results from spectral characterization of regenerated photoprotein show a maximum emission wavelength at 489nm, and spectra do not indicate bimodal distribution as was previously observed. Characterization and site‐directed mutagenesis of the luciferin binding site of the Malpighian tubules luciferase‐like enzyme from Zophobas morio (Coleoptera: Tenebrionidae) RA Pradoa,c, JA Barbosab and V Veviania,c aLaboratory of Biochemistry and Biotechnology of Bioluminescence bGraduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCAR), Sorocaba, SP, Brazil cGraduate Program of Evolutional Genetics and Molecular Biology, São Carlos, SP, Brazil E‐mail: pradorogilene@yahoo.com.br Previously, we cloned the first functional luciferase‐like enzyme from Zophobas morio giant mealworm, a non‐bioluminescent Coleoptera. This enzyme belongs to AMP‐CoA‐ligase superfamily and despite being distantly related to luciferases it displays a weak luminescent activity at the presence of D‐Luciferin and MgATP making it a good protoluciferase model. The cDNA was subcloned in pColdII vector and the enzyme highly expressed (10 mg/L culture) allowing the purification and characterization of the protein. As expected, the Km for luciferin is much higher than that of beetle luciferase, since luciferin is not the natural substrate of this enzyme, however the Km to ATP is similar to that of beetle luciferases and other AMP‐ligases. Multialignment of amino acid sequence of this protoluciferase and beetle luciferases showed several substitutions of otherwise conserved residues in the luciferin binding site. A site‐directed mutagenesis survey of the luciferin binding site showed that most of the replacements of protoluciferase residues by those found in beetle luciferases drastically reduced the luminescent activity. Among them, the mutation I205R (R218 in Photinus pyralis) resulted in a bathchromic shift in the emission spectrum and increased the affinity to luciferin, probably, as a consequence of increasing the polarity around the oxyluciferin phenolate, despite of reducing the activity. On the other hand, the mutation I327T resulted in 2‐fold increase of luminescent activity, a 15 nm hypsochromic shift and broadening of the emission spectrum. This position is inserted in the important β‐hairpin motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase), and the corresponding residue T/S345 in beetle luciferases makes stabilizing hydrogen bonds, that may help to stabilize the loop in a catalytic conformation. We suggest that the substitution I327T could have been critical for the evolution of efficient bioluminescence in beetle luciferase. Although the natural substrate and biological function of this enzyme has not been identified yet, we suspect the participation of this enzyme in excretion and detoxification of xenobiotics. Financial support: FAPESP (2007/07950‐3) and CNPq, Brazil. Bioluminescent beetles in the Atlantic Rain‐Forest and transition from Cerrado to the Amazon forest: biodiversity, bioprospection and use in bioindication RA Pradoa,c, R Machadoa,b, O Hagena, MY Rochaa, DT Amarala,c and VR Viviania,c aLaboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring bGraduate Program on Biological Diversity and Conservation, Federal University of São Carlos (UFSCar), Sorocaba, Brazil cGraduate Program of Evolutional Genetics and Molecular Biology, São Carlos, SP, Brazil. E‐mail: biota.biolum@hotmail.com Among terrestrial organisms, bioluminescence occurs predominantly in the order Coleoptera (beetles), which accounts with about 2000 described species distributed around the world in the superfamily Elateroidea, which include the families Lampyridae (1800), Phengodidae (150) and Elateridae (100). Besides being extremely important sources of luciferases and reporter genes for bioanalytical purposes, bioluminescent beetles are recently starting to be used as environmental indicators. Most species occur in the Neotropical region, with Brazil hosting the largest number of species, mainly in the Atlantic rain‐forest, Cerrado (Savannas) and the Amazon forest. The two former ecosystems are the most threatened ones. The program Biota‐Biolum is investigating the Brazilian diversity of luminescent beetles for molecular evolution studies, conservation and bioprospection purposes. Data were obtained during the past 20 years in the Atlantic rain forest of São Paulo state and Cerrado from Central‐west Brazil near Parque Nacional das Emas (Goiás state). A total of 49 different species were catalogued from the Atlantic rain forest (Lampyridae: 35; Phengodidae: 6; Elateridae: 7; Staphylinidae: 1). Among the Atlantic rain forest investigated areas, the preserved Biological station of Boraceia (Salesópolis), accounted with the largest number of species (30), being considered a hot spot, followed by Campinas (25), Rio Claro (22) and Sorocaba (20). In Central‐west Cerrado, 27 species were found (Lampyridae: 13; Phengodidae 9; Elateridae: 4; Staphylinidae: 1). The cerrado ecosystem includes some unique and severely threatened species such as the larval click beetle Pyrearinus termitilluminans that is responsible for the luminous termite mounds phenomenon, and rare types of railroadworms, that already provided important luciferases for bioanalytical purposes. During the last 20 years the cerrado around Parque Nacional das Emas was totally overtaken by soy monoculture, and the few remnant areas were severely impacted, with disappearance of several species of railroadworms. More recently we expanded our studies to the Araguaia's river basin around Bananal Island, in a transition ecosystem between Cerrado and Southeastern Amazon forest. Finally, the impact of artificial night lighting in the occurrence and activity of fireflies is being evaluated in the urban areas of Sorocaba and Campinas. These studies are showing that the diversity of species quickly shrinks around illuminated areas, whereas a few ones may persist. Besides the scientific and biotechnological importance, bioluminescent species may constitute new important environmental bioindicators, mainly for assessing the nocturnal environments, water courses and marshy environments. (Financial support: Fundação de Amparo a Pesquisa do Estado de São Paulo FAPESP‐Biota: 0651911‐0, Brazil). Theoretical study of the spectral‐structural relations of a series of Oxyluciferin Derivatives Xue Qin Ran* and John D. Goddard Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1 *Presenting author. Tel: (+1) 519‐824‐4120. Fax: (+1) 519 766 1499. E‐mail: xran@uoguelph.ca The absorption and emission spectra of a series of oxyluciferin derivatives in Keto‐Enol‐Enolate and Phenol‐Phenolate forms as well as with different substituents were systematically investigated (Figure 1, 2). The roles of the neutral and ionized 6′‐OH, 6′‐NR2 and 6′‐OCH3 groups were considered. The ground state geometries were optimized by M06HF/6‐31+G(d) and the absorption spectra were calculated by TDDFT//B3LYP/6‐31+G(d). The excited state geometries and the emission spectra were studied by TDDFT//B3LYP/6‐31+G(d). It was demonstrated that a wide spectral range of emission color can be obtained by these various oxyluciferin derivatives. Some of these species are newly investigated with computational methods. Figure 1. The structures of 6′‐Methylaminooxyluciferin (MNHOxyLH2), Cyclic Aminooxyluciferin (CNHOxyLH2), Cyclic Methylaminooxyluciferin (CMNOxyLH), 6′‐Methoxy‐5,5‐dimethyloxyluciferin (MDMOxyLH), 5,5‐Dimethyloxyluciferin (DMOxyLH) and 4‐Dehydroxy‐5,5‐dimethyloxyluciferin (DHDMOxyLH). Figure 2. Chemical Equilibria of 6′‐Dehydroxyoxyluciferin (DHOxyLH), 6′,6′‐Dimethylamino‐oxyluciferin (DMNOxyLH),5‐Methyloxyluciferin (MOxyLH2). Some investigations on the chemical origin of the multicolor firefly bioluminescence Ai‐Min Rena, Chun‐Gang Mina and John D Goddardb aState Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, P. R. China bDepartment of Chemistry, University of Guelph, Guelph, Ontario, Canada Fireflies naturally emit multicolor light from green (≈530 nm) to orange, and even to red (≈635 nm). In order to explain the variation in the color of the bioluminescence, many hypotheses have been proposed to date. However, there is still no consensus on which hypothesis best describes the mechanism behind the multicolor bioluminescence. The relationship between the wide range of bioluminescent colors and the structure of the light emitter remain challenging problems. Experimental studies of the light emitters are hindered due to the extreme instability of OxyLH2. Thus theoretical predictions preferably with ab initio methods are advantageous. In this section it is reviewed that all available theoretical data of us are used to study aspects of the six hypotheses regarding the OxyLH2‐based light emitters from fireflies. Light emitting system in a deep sea shark: Etmopterus spinax (Squaloidea: Etmopteridae) M. Renwart and J. Mallefet (Marine Biology Laboratory, Earth and Life Institute, UCL, Belgium) The biochemistry of light emitting systems has been largely studied in invertebrates. Among vertebrates, only fishes are endowed with this capability and the mechanism of light emission has merely been investigated in bony fishes. Although less known, because rather rare and difficult to observe, cartilaginous fishes also contain bioluminescent members. Two families are in concern, Dalatiidae and Etmopteridae1, information about the biochemistry of their luminous system are lacking. In this work, we aim to describe for the very first time the chemiluminescent reaction involved in a shark species: Etmopterus spinax (Etmopteridae). E. spinax is a deep‐sea species displaying a continuous blue luminescence on its ventral and lateral faces2. Classical cross‐reactions with known luminous substrates, such as known imidazolopyrazines, do not produce light per se, suggesting a new luminous system in this species. Studies are now in progress to detect and purify the substrate of the reaction by biochemical methods. This work was supported by an FRS‐FNRS grant to M. Renwart. J. Mallefet is a researcher associate of the FRS‐FNRS. Hubbs CL, Iwai T, Matsubara K. External and internal characters, horizontal and vertical distributions, luminescence, and food of the dwarf pelagic shark Euprotomicrus bispinatus. Bull. Scripps Inst. Oceanogr. 1967;10:1–64. Claes JM, Mallefet J. Early development of bioluminescence suggests camouflage by counterillumination in the velvet belly lantern shark Etmopterus spinax (Squaloidea: Etmopteridae). J. Fish Biol. 2008;73:1337–50. Bioluminescent assays for monitoring of air pollution NV Rimatskayaa, EV Nemtsevaa,b and VA Kratasyuka,b aSiberian Federal University, 79 Svobodny Prospect, Krasnoyarsk, 660041, Russia bInstitute of Biophysics SB RAS, 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia E‐mail: Shmanko_Nadya@mail.ru When compared with the other available tests measuring water toxicity the bioassay methods based on the luminescent bacteria, soluble and immobilized coupled system of NAD(P)H:FMN‐oxidoreductase‐luciferase (reagent “Enzimolyum”) have certain advantages as for analysis speed, handling and cost. However the available bioluminescent methods were not yet applied to measure air pollution. So we aimed to determine the sensitivity of luminous bacteria and their enzymes to air samples differed by industrial pollution degree. Air samples were collected in the clean (Akademgorodok, sample#1) and polluted (the coal power plant area, sample#2) districts of Krasnoyarsk city. The air samples were collected into liquid absorption medium (water, ethanol or acetone). The standard aspirating device performing 1.0 liter per minute was used. Chemical composition of the samples was analyzed with gaseous chromatograph (Agilent Technologies 7890A). To compare the sensitivity of assays the numbers of dilution of the samples necessary to remove toxic effect were considered. Results are presented in the table 1. Table 1. Comparative characteristics of bioluminescent assays used to monitor air pollution Type of bioassay Soluble coupled system Luminescent bacteria Immobilized coupled system (Enzimolyum) Number of components (simplicity) 5 2 3 Duration of assay, min 10 5–30 7 Sensitivity (number of dilutions), sample#1 / sample#2 Water 2000 / 3 700 / 3 16000 / 3 Ethanol 700 / 1 1 / 1 250 / 3 Acetone 2000 / 3 1 / 1 >2000 / 3 Storage conditions 2 months at +15°C, 3 years at –18°C 6 months at +5°C, 1 year at –18°C 2 years at +4 ‐ +25°C The results indicate that water is the better than ethanol or acetone medium for air sample preparation because of its sufficient capacity to absorb organic compounds, absence of interfering effects on bioluminescent. The sensitivity of soluble and immobilized enzymes is 3–24 times higher than sensitivity of bacterial‐based test. The immobilized reagent provides the reduction of the time required to complete the analysis (down to 7 minutes), easy‐to‐use, higher sensitivity (allowed dilutions is up to 16000), possibility to increase the volume of the sample up to 97% of the total one. Thus we showed the possibility to apply the bioluminescent bioassays based on immobilized reagent " Enzimolyum" for air pollution monitoring. This work was supported by the Federal agency of science and innovations (contract No 02.740.11.0766), the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058). Emission modulations in the bioluminescent firefly luciferase‐oxyluciferin system studied by molecular dynamics on the electronically excited potential energy surface Chang‐ik Song and Young Min Rhee* Department of Chemistry, Pohang University of Science and Technology (POSTECH) Pohang, 790‐784 Korea Dynamics of the Japanese firefly (Luciola cruciata) luciferase‐oxyluciferin complex in its electronically excited state is studied using various theoretical approaches. We have focused on mimicking the physiological conditions with realistic models of the chromophore oxyluciferin and the luciferase enzyme together with solvating water molecules. For the chromophore, quantum chemical calculations are utilized to optimize the force field parameters in terms of the intramolecular and intermolecular potential terms (Figure 1). With the potential energy expressions, we have first performed molecular dynamics simulations of the complex starting from various different initial conditions and revealed that the chromophore‐surrounding interaction patterns differ rather severely in the excited state compared to the situations in the ground state. Most notably, ion pair formations were found to be important in the exicted state but not in the ground state. Such ion pairs play stabilizing roles for the chromophore, and the difference between the ground and the excited states is mainly caused by the charge transfer nature of the electronic transition. Figure 1. Changes of the Lennard‐Jones parameters of oxyluciferin after the electronic transition. (a) Differences in atomic ε‐parameters: brighter parts denote that the dispersive potential wells of the atoms become shallower in the excited state. (b) Differences in σ‐parameters: darker parts exhibits that the potential wells become wider in the excited state. Overall, the benzothiazole and thiazole ring moieties show opposite trends. Along the trajectories, we have also sampled different ligand‐protein conformations and then applied the quantum‐mechanicsmolecular‐mechanics (QM/MM) approach to investigate the molecular driving force for tuning the emission color. We show a close relationship between the emission color variation and the environmental dynamics, mostly through electrostatic effects from the chromophore‐surrounding interaction. Quite naturally, charged residues which are adjacent to the oxyluciferin are relatively important. However, the overall protein effect and the structural fluctuations exert collective modulation effect on the emission color (Figure 2). Figure 2. The correlation between the emission energy and the electrostatic potential (ESP) difference on the two terminal oxygen atoms of oxyluciferin. The ESPs were calculated by considering all protein atoms. We also discuss the importance of considering time scales of the luminescence and the dynamics of the interaction. Interestingly, the ion pair formation dynamics is observed to be rather fast compared to conventional fluorescence lifetimes while the resolvation around the oxyluciferin shows a mixed behavior when it is bound in the protein. References Song C‐I, Rhee YM. Development of force field parameters for oxyluciferin on its electronic ground and excited states. Int. J. Quantum Chem. 2011;111:4091–105. Song C‐I, Rhee YM. Dynamics on the electronically excited state surface of the bioluminescent firefly luciferase‐oxyluciferin system. J. Am. Chem. Soc. 2011;133:12040–9. WARNING: The light‐emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same! Daniel Roca‐Sanjuána,*, Mickael G. Delceya, Isabelle Navizetb, Nicolas Ferréc, Ya‐Jun Liud and Roland Lindha aDepartment of Chemistry ‐ Ångström, Theoretical Chemistry Programme, Uppsala University, P.O. Box 518, S‐75120 Uppsala, Sweden bMolecular Science Institute School of Chemistry, University of the Witwatersrand, PO Wits Johannesburg 2050 South Africa cUniversités d'Aix‐Marseille I, II, et III‐CNRS UMR 6264: Laboratoire Chimie Provence, Equipe: Chimie Théorique Faculté de St‐Jérome, Case 521, 13397 Marseille Cedex 20, France dKey Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China *Corresponding author. E‐mail: Daniel.Roca@kvac.uu.se The general scenario in a great amount of chemiluminescence and most of the bioluminescence reactions consists on a thermally activated decomposition reaction of a dioxetanone‐based molecule. In this process, an excited state species is produced and a carbon dioxide group released. Figure 1. Comparison of the chemiluminescent (left) and fluorescent (right) intrinsic properties obtained in the quantum‐chemical investigations on a small model for coelenteramide and Cypridina oxyluciferin. From top to bottom: electronic excitation which characterizes the excited state, geometrical parameters of the light emitter, general profile for the potential energy surfaces of the ground and excited states, and strategies that lead to both sets of findings. Typical investigations on the characterization of the light‐emitting species focus on the decomposition product. In particular, the emission spectrum of this compound after irradiation with light is employed in some experimental works to obtain chemiluminescence properties. Meanwhile, geometry optimizations of the excited state are performed in some theoretical studies by using the product as starting point. In the present work,1 we apply the same computational procedure (strategy 1 in Figure 1) with highly accurate quantum‐chemical methods to optimize the excited state of small models of the coelenteramide and Cypridina oxyluciferin systems, which are the light‐emitting species in some marine bioluminescent organisms, such as the hydromedusa Aequorea and the hydroid Obelia, in the first case, and the ostracod Vargula hilgendorfii, in the latter. In a second step, we also carry out excited‐state geometry optimizations starting from a geometry closer to the transition state structure and the region of the potential energy surfaces where the system populates the excited state (strategy 2 in Figure 1). Two different light‐emitting structures are found with significant differences at the molecular level –electronic structures and molecular geometries– and in the emission energies. While the first excited‐state equilibrium minimum corresponds to the fluorescent state of the molecule, the last finding can be associated to the chemiluminescence phenomenon. On the basis of these results some recommendations are given here for future designs of experimental and theoretical procedures to investigate the chemiluminescence and bioluminescence phenomena. Reference 1. Roca‐Sanjuán D, Delcey MG, Navizet I, Ferré N, Liu Y‐J, Lindh R. J. Chem. Theor. Comp. 2011, DOI: 10.1021/ct2004758 NEW TOOLS FOR MULTIPLEXED BIO‐CHEMILUMINESCENT BIOSENSORS: METABOLICALLY BIOTINYLATED THERMOSTABLE RED‐ AND GREEN‐EMITTING PHOTINUS PYRALIS LUCIFERASES Aldo Rodaa,b*, Chiara Canalia, Elisa Michelinia,b, Luca Ceveninia, Luisa Stella Dolcia, Patrizia Simonic and Bruce R. Branchinid aDepartment of Pharmaceutical Sciences, University of Bologna, Italy bINBB, Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy cDepartment of Clinical Medicine, University of Bologna, Bologna, Italy dConnecticut College, New London, CT, USA *E‐mail: aldo.roda@unibo.it Thanks to their high detectability, bio‐chemiluminescence (BL‐CL) based probes are very appropriate for ultrasensitive multiplex biosensing using nucleic acids or antigen‐antibody molecular recognition.1 BL‐CL probes can be easily implemented in miniaturized biosensor devices for new generation diagnostics able to measure in a single run a wide panel of different disease‐specific biomarkers.2,3 Multiplex imaging detection can be achieved by using a sensitive CCD camera and by i) spatially resolving the light emitted by different probes, ii) sequentially measuring the light signals triggered by different substrates (e.g., horseradish peroxidase, HRP, and alkaline phosphatase, AP) and iii) spectrally resolving signals obtained from labels emitting at different wavelengths (e.g., different luciferase mutants).4 Two new metabolically synthesized biotinylated luciferases were produced to obtain ultrasensitive universal BL probes. We selected two mutants from P. pyralis luciferase, PpyRE10 and PpyGRt9, emitting in the red and green regions of the visible spectrum with improved thermostability when compared to widely used CL‐BL systems such as HRP/luminol/enhancer, AP/dioxetane phosphate and G. princeps luciferase/coelenterazine. Thermostable red‐ and green‐emitting mutants were obtained by site‐directed mutagenesis.5 cDNA sequences encoding for luciferase mutants were cloned downstream to an optimized version of K. pneumoniae oxaloacetate decarboxylase biotinyl‐binding domain containing a lysine to which biotin prosthetic group is in vivo covalently bound. High levels of biotinylation were achieved by using an inducible vector for biotin holoenzyme synthetase (BirA) overexpression in E. coli BL21(DE3) strain. Biotinylated luciferases were purified by affinity chromatography and stored at + 4 °C for up to 5 months without loosing activity. They showed narrow emission bandwidths and 69 nm separation between the two λmax (617 and 548 nm, respectively), thus holding great promise for new multiplex applications. Thermal and pH stability assays revealed high stability at 25–42 °C for up to 6 hours (e.g., half‐life at 37 °C, pH 7.8 was 10 hours for the green‐emitting mutant). To compare the analytical performance of these newly developed BL probes with commercially available ones, the probes were captured by streptavidin‐coated black microtiter plates and dose‐response curves were obtained. Probe Substrate LOD Biotinylated PpyGRt9 D‐Luciferin, MgATP 7 x 10‐13 mol Biotinylated PpyRE10 D‐Luciferin, MgATP 2 x 10‐12 mol Biotinylated G. princeps luciferase Coelenterazine (native) 4 x 10‐15 mol Biotinylated HRP Luminol/enhancer 2 x 10‐12 mol Biotinylated AP Dioxetane phosphate 3 x 10‐15 mol The range of linearity for all the studied probes extended at least 5 orders of magnitude over the LOD. Sequential assays of different target analytes in the same tube or spot were performed without significant substrate incompatibility between P. pyralis mutants and G. princeps luciferase and using AP or HRP substrate at the end of the sequential reading. Therefore, BL‐CL biotinylated probes are universal tools suitable for miniaturized biosensors and assisted microfluidics can be used to properly deliver substrate(s) even in the same spot containing different probes. Thus, multiplexed analysis can be achieved by spatial resolution (e.g., by performing contact imaging detection with a CCD sensor) or spectral resolution of the analytical signals. Roda A, Guardigli M. Anal Bioanal Chem. In press, DOI: 10.1007/s00216‐011‐5455‐8 Roda A, Mirasoli M, Dolci LS, Buragina A, Bonvicini F, Simoni P, Guardigli M. Anal Chem. 2011;83(8):3178–85. Roda A, Cevenini L, Michelini E, Branchini BR. Biosens Bioelectron. 2011;26(8):3647–53. Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini B, Roda A. Anal Chem. 2008;80(1):260–7 Branchini BR, Ablamsky DM, Rosenberg JC. Bioconjug Chem. 2010;21(11):2023–30. Synthesis and optical characterization of nanoparticles with persistent luminescence in the red‐near infrared range Céline Rostichera,*, Corinne Chanéaca, Bruno Vianab and Aurélie Bessière b *E‐mail: celine.rosticher@etu.upmc.fr aLaboratoire Chimie de la Matière Condensée de Paris ‐ Université Pierre et Marie Curie ‐ UMR CNRS 7574 ‐ Collège de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France bLaboratoire Chimie de la Matière Condensée de Paris ‐ Université Pierre et Marie Curie ‐ UMR CNRS 7574 –Ecole Nationale Supérieure de Chimie de Paris – 75005 Paris, France In the past decades, there has been a great improvement in the domain of the imaging systems and new imaging tools have been developed. But commercialised techniques are still very expensive and potentially harmful(1). Thus optical imaging, in which photons are the information source, is a rapidly expanding field. In vivo imaging implies some problems due to the biological environment, such as the tissues' autofluorescence(2) and absorption(3). Luminescent markers have to emit in the wavelength therapeutic window because tissues absorb UV and visible light and only a red‐near IR emission can go through the tissues(3). Therefore we have recently developed inorganic luminescent nanoparticles (NPs) suitable for in vivo imaging and that can master the difficulties due to the biological environment. The NPs are first excited by a UV light for a few minutes outside the animal, then injected to the animal, where they emit visible light for hours after the injection (Figure 1). Autofluorescence, resulting from external illumination during signal acquisition, is therefore avoided. Figure 1. a) Transmission electronic microscopy image, b) In vivo experiment principle and in vivo images. Our starting material composition for the silicate compounds was Ca0.2Zn0.9Mg0.9Si2O6 developed by Le Masne de Chermont et al. (4). These new long luminescent NPs emit in the red‐near infrared range and the emission can last for several hours. But there are some limitations to these compounds: the luminescence intensity has to be increased, the NPs' size has to be decreased and Zn2+ incorporated in the matrix induces the coexistence of two phases. Thus, we chose to develop CaMgSi2O6: Eu2+, Dy3+, Mn2+ compounds as new long luminescent NPs. Those are synthesized by two sol gel methods: nucleation of NPs in a silica gel and nucleation of NPs in silica colloids (Stöber process). In the aim of improving their luminescent properties, we have studied the influence of the chemical composition on the luminescence: Ca/Mg/Si molar ratio, TEOS and the doping elements(5) and ratio. Persistent luminescence spectra of CaxMgySi2O6 (with x and y smaller than 1) compounds doped Eu2+ (0.5%), Dy3+ (1%), Mn2+ (2.5%) materials showed three emission bands after a UV irradiation at 350 nm: a first one at 480 nm corresponding to Eu2+ emission and two bands at 580 and 680nm corresponding to Mn2+ emission (figure2). We observed that the more the Ca/Mg molar ratio increases, the more the intensity of Eu2+ and Mn2+ bands decreases, causing a strong decreasing of the persistent luminescence intensity in these compounds. Figure 2. Persistent luminescence spectrum of CxMyS2O6: Eu2+ (0.5%), Dy3+ (1.0%), Mn2+ (2.5%) ‐ λexc = 254nm for 2min. We study gadolinium oxysulfides doped by some luminescent ions such as Eu2+, Ti4+, Mg2+ which are interesting bimodal sensors with two properties that can be coupled: luminescence and magnetism. Gadolinium compounds(6) are already used in clinical medicine as MRI contrast agents, used to assist in the visualization of blood vessels. The gadolinium oxysulfides are synthesized by hydrothermal method, this permits us to obtain NPs, essential for in vivo imaging. The final objective of this work is to obtain biocompatible and/or biodegradable NPs with the highest luminescence intensity and time, therefore we want to develop NPs of doped calcium phosphates, which are the most important inorganic constituents of biological hard tissues. All these compounds were characterized by TEM, XRD, and their luminescent properties were studied with a CCD camera coupled with a spectrometer for spectral analysis. References Cherry SR. Annu. Rev. Biomed. Eng. 2006;8:35. Frangioni JV. Curr. Opin. Chem. Biol., 2003;7:626‐34. Cheong WF, Prahl SA, Welch AJ, IEEE J. Quantum Electron. 1990;26:2166‐85. Le Masne de Chermont Q. Proc. Natl. Acad. Sci. USA 2007;104:9266‐71. Maldiney T, Lecointre A, Viana B, Bessiere A. J. Am. Chem. Soc. 2011;133:11810–15. Fortin MA, Petoral R, Soderlind F. Nanotechnology 2007;18:395501. Effects of type of binder and conducting phase on performance of solid state electrochemiluminescence composites A Safavia,b*, F Sedaghatia and H Shahbaazic aDepartment of Chemistry, College of Sciences, Shiraz University, Shiraz, 71454, Iran bNanotechnology Research Institue, Shiraz University, Shiraz, Iran cChemistry Department, University of Calgary 2500 Uni. Dr. NW, Calgary AB T2N1N4, Canada *E‐mail: safavi@chem.susc.ac.ir Different immobilization processes are developed for modification of electrodes with active luminescent materials. ECL of Ru(bpy)32+ has received much attention due to its superior properties including high sensitivity and stability under moderate conditions in aqueous solutions.1 Because of noteworthy properties of ionic liquids (ILs), it is important to introduce IL in to ECL study. Applications of ionic liquids as the binder in place of traditional oil for constructing carbon paste electrodes (CPE) have been reported for ECL study in recent years.2 Although the entire reported ECL sensors confirmed improved ECL characteristics, however, pretreatment of the electrodes or the fabrication procedures of the modified electrodes for solid state ECL are somewhat difficult. In the case of Ru(bpy)32+, it is hydrophilic and dissolves simply in aqueous solutions, causing the problem of leaching. Therefore, new materials and immobilization methods are still required in order to improve both the sensitivity and the long‐term stability of ECL‐based sensors. Excellent properties of carbon ionic liquid electrode3 (CILE) provided us an idea to apply it for construction of a solid state ECL sensor. In this survey, different types of solid state ECL sensors such as Ru‐graphite/OPPF6, Ru‐graphite/BMIMPF6 and Ru‐CPE were fabricated and their performances were evaluated. Performances of paraffin oil and two ILs as the binders were compared for construction of solid state ECL. Ru‐CPE was not a good choice to apply as a soild state ECL sensor owing to leakage of luminophor in to the electrolyte. The ECL behavior of Ru‐graphite/OPPF6 electrode has been studied. At E = 1350 mV, one image from the surface of the electrode was captured at an exposure time=30 s (Fig 1). The electrochemical and ECL behavior of Ru‐graphite/OPPF6 and Ru‐graphite/BMIMPF6 were investigated and compared (Fig. 2). Simple preparation, low background current, fast electron transfer rate, low cost and long‐term stability were attractive properties of Ru‐graphite/OPPF6 composite electrode. Also, for study of the type of the conducting phase, graphite was substituted by MWCNTs and Ru‐MWCNT/OPPF6 was constructed. ECL and electrochemical activities of this composite were compared with those of Ru‐graphite/OPPF6. Figure 1. a) Original image and b) its recolor of electrode surface (Ru‐graphite/OPPF6) at 1350 mV and EX = 30 s in PBS pH = 7 in the presence of 10 mM TPrA. Figure 2. ECL signal vs. time for the composite of a) Ru‐graphite/OPPF6 b) Ru‐graphite/BMIMPF6 in the presence of 1 μM TPrA (background signal of ECL was subtracted). References Hu L, Xu G. Chem. Soc. Rev. 2010;3275. Dai H, Wang Y, Wu X, Zhang L, Chen G. Biosens. Bioelectron. 2009;24:1230 Maleki N, Safavi A, Tajabadi F. Anal. Chem. 2006;78:3820. Plant peroxidases‐catalyzed detection systems and their use in ultrasensitive chemiluminescent enzyme immunoassay Ivan Yu. Sakharov and Marina M. Vdovenko Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119991, Russia E‐mail: sakharovivan@gmail.com The most sensitive format of enzyme immunoassay (EIA) is the assay with chemiluminescent (CL) detection of enzyme activity of immunoreagents. Traditionally in CL‐EIA horseradish peroxidase (HRP) and 4‐iodophenol (PIP) are used as enzyme label and enhancer, respectively. This enhanced chemiluminescence reaction (ECR) based on luminol oxidation has been successfully used in development of ultra‐sensitive immunochemical kits for the determination of various compounds. Main drawbacks of the method of peroxidase activity measurement using ECR with HRP/PIP are the relatively quick decay of CL signal and its insufficiently high sensitivity. A replacement of PIP with 3‐(10′‐phenothiazinyl)propane‐1‐sulfonate (SPTZ) as the enhancer allowed to increase the intensity of chemiluminescent signal, the light intensity being practically unchanged for a long time. Moreover, a combination of SPTZ and 4‐morpholinopyridine (MORPH) allowed additionally the increase of CL no affecting the kinetics of CL decay. The optimization of the experimental conditions for ECR catalyzed by soybean (SbP) and horseradish peroxidases was carried out by a 25 full factorial design. In the case of use of SPTZ/MORPH system a detection limit of SbP was 0.03 pM that was 40‐fold lower than that of HRP/PIP system. The SbP/SPTZ/MORPH detection system was applied successfully in construction of ultrasensitive EIA kit for determination of thyroglobulin in human serum. The study showed that a lower detection limit (LOD) of the CL‐EIA with SbP/SPTZ/MORPH was 10 times lower than in the assay with HRP/PIP. The obtained results open good perspectives for use of ECR with SPTZ/MORPH in the development of ultra‐sensitive immunoassay kits. Synthesis and chemiluminescence of N‐(5‐halogen‐2‐oxo‐2,3‐dihydrobenzofuran‐3‐yl)benzamides Stefan Schramm, Dieter Weiß and Rainer Beckert Friedrich Schiller University – Jena, Humboldtstr. 10, Jena, 07743, Germany For the first time it was possible to prepare successfully compounds of the class of the N‐(5‐halogen‐2‐oxo‐2,3‐dihydrobenzofuran‐3‐yl)benzamides (NHDB) (Fig. 1). After the addition of a strong base like 1,8‐Diazabicyclo‐(5.4.0)undec‐7‐ene (DBU) in an polar aprotic solvent like acetone, THF, DMF, acetonitrile or dibutylphthalate they indicated a surprising intensive amount of chemiluminescence (Fig. 2). This induced us, based on the work of Lofthouse et al. (1) and Matuszczak (2) which studied congeneric compounds, to explore the class of the NHDB more intensively. In the progress of research we started our synthesis with the reaction of alpha‐Hydroxyhippuric acid and the appropriate p‐Halogenphenol which resulted in the 2‐Benzamido‐2‐(5‐halogen‐2‐hydroxyphenyl)aceticacids (BHHA). In the next step we converted the BHHAs with acetic anhydride into the corresponding NHDBs. Quantitative chemiluminescence studies indicate an increase of the amount of chemiluminescence from the bromine to the fluorine derivate, although the wavelength at the point of the maximum emission is nearly the same. Figure 1. Figure 2. References Lofthouse GJ, Suschitzky H, Wakefield BJ, Whitetaker RA, Tuck B. J. Chem. Soc. Perkin Trans. I, 1979;1634. Matuszczak B. Monatsh. Chem. 1996;127:1291. Fusion proteins of Luciola mingrelica firefly luciferase. Preparation, properties, application DV Smirnova, MI Koksharov and NN Ugarova, Dept. of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia E‐mail: S_mir_nova@mail.ru Currently there is a necessity for new bioanalytical highly sensitive and highly specific reagents for the detection of nano‐quantities of different physiologically active substances and pathogenic germs. One of the widely used approaches to develop such systems is the use of fusion proteins that combine the high sensitivity of the enzyme label and the high specificity of the protein, which is able to bind the specific target. This leads to the fixation of the enzyme label on the surface of the target. Different luciferases can be used as an enzyme label, particularly firefly luciferase (1–2), due to the high quantum yield of its bioluminescent reaction leading to the high sensitivity of the bioluminescent detection, low background signal owing to the high stability of the substrate and the simple procedure of expression and purification of the protein in required quantity. Streptavidin or biotin‐binding domain can be used as a selective component because of their high binding constant. The four plasmids were constructed by methods of genetic engineering, those encode fusion proteins incorporating the thermostable mutant of Luciola mingrelica firefly luciferase (luc4TS) (3), and biotin carboxyl carrier protein domain (bccp87) or streptavidin (SA). The structures of plasmids are shown at Fig. 1. All fusion proteins were expressed as his6‐forms and purified using metal‐chelate chromatography. Three plasmids coding luciferase‐streptavidin fusions differed by mutual location of the luciferase and streptavidin genes and the linker composition. As a result the luciferase activities of the fusion proteins were different. The fusion proteins Luc4TS‐link7‐SA‐his6, SA‐link5‐Luc4TS‐his6 and SA‐link28‐Luc4TS‐his6 accounted for 15, 20, 45% of 4TS luciferase activity respectively, whereas the activity of luc_4TS‐bccp87 accounted for 60% of 4TS activity. Figure 1. Structures of plasmids coding fusion protein luciferase‐straptavidin and luciferase‐biotin binding protein. Catalytic properties, thermostability and bioluminescence spectra of the fusion proteins were found to be close to that of the non‐fused luciferase. Linear relationship was observed between luciferase activity and concentration of fusion proteins till 0.1 pmol/L. The ability of the SA‐Luc4TS fusions to bind biotin was shown with use of the biotin‐conjugated bovine serum albumin. We have shown that streptavidin‐«biotinylated luciferase» complex can be used in ELISA for the determination of Salmonella typhimurium cells in the range from 104 to 5·106 CFU/ml. References Karp M, Oker‐Blom CA. streptavidin–luciferase fusion protein: comparisons and applications. Biomol. Eng. 1999;16:101–4. Nakamura M, Funabashi H, Yamamoto K, Ando J, Kobatake E. Construction of streptavidin‐luciferase fusion protein for ATP sensing with fixed form. Biotechnol. Lett. 2004;26:1061–6. Koksharov MI, Ugarova NN. Thermostabilization of firefly luciferase by in vivo directed evolution Protein Eng., design and selection. 2011;24:835–44. Chemiluminescent system of bioobjects antioxidant activity definition Dmytro V. Snizhko and Mykola M. Rozhitskii Laboratory of Analytical Optochemotronics, Kharkiv National University of Radio Electronics,14 Lenin Ave, 61166, Kharkiv, Ukraine; E‐mail: rzh@kture.kharkov.ua The work includes theoretical and experimental investigation, aimed at development of chemiluminescent (CL) method intended for human being antioxidant system study that includes oxidation processes of biological organic compounds and activation of antioxidant system under condition of active forms of oxygen influence. There are modified physical and mathematical models of peroxide lipids oxidation processes that accounts for presence of antioxidants and variation of their amount in bioobject under influence of exogenous active forms of oxygen including case of ozone therapy and allows predicting therapeutic influence of active forms of oxygen on the patient's organism. For the first time it was theoretically and experimentally proven that rate and kinetics of active forms of oxygen in biosamples obtained under influence one of active oxygen form–ozone—can be determined by means of induced CL kinetics study. There was developed automated software and hardware analytical chemiluminescent system that accomplishes chemiluminescent assays investigation of active form of oxygen influence on biological objects antioxidant system state. Also there was developed chemiluminescent method of rapid assay of characteristics of patient's antioxidant activity during therapy using active forms of oxygen with microvolumes of biosample (blood or urine). The author expressed gratitude for STCU (project 5067) for financial support (Project Manager – Prof. M.M. Rozhitskii). Fluorescence studies of thermal affect on enzymes of coupled enzymatic system of luminous bacteria NADH:FMN‐oxidoreductase‐luciferase in viscous media IE Sukovataya, OS Sutormin and VA Kratasyuk Siberian Federal University, Krasnoyarsk, Russia E‐mail: ISukovataya@sfu‐kras.ru The protein—solvent interaction is a general problem concerning the understanding of enzyme catalysis mechanisms. In earlier reports (1), we described the effects of organic solvents on catalytic activity of bacterial luciferases. It is known that tryptophan fluorescence of proteins is sensitive to changes of physical‐chemical property of environment. The emission spectrum of indole group of tryptophan is highly sensitive to solvent polarity. The emission of indole may be blue shifted if the group is buried within a native protein, and its emission may shift to longer wave‐lengths (red shift) when the protein is unfolded. Thus, studying the fluorescence of luciferase from Photobacterium leiognathi and NAD(P)H:FMN‐oxidoreductase from Vibrio harveyi (2) in the presence of viscous solvents upon heating at 35°C could investigate the effects of viscous solvents and temperature on the tertiary and secondary structural changes of luciferase and NAD(P)H:FMN‐oxidoreductase. The results of fluorescence studies shown that the enzymes in the absence of osmolytes had maximum of the fluorescence intensity at higher temperature, suggesting that temperature increment cause changes in the tertiary structure of protein resulting in the exposure of buried aromatic residues to the solvent. In bacterial luciferase and NAD(P)H:FMN‐oxidoreductase are present 7 and 4 indole groups of tryptophan, respectively. In the presence glycerol and sucrose it was shown that increasing concentration of osmolytes lead to a monotonic decrease of fluorescence intensity of luciferase and NAD(P)H:FMN‐oxidoreductase due to concentration quenching of external tryptophan. In the presence of sucrose has been registered blue shifted of the fluorescence spectra of the enzymes. Fluorescence studies of thermal affect on enzymes of coupled enzymatic system of luminous bacteria NADH:FMN‐oxidoreductase‐luciferase in viscous media showed the absence of luciferase and NAD(P)H:FMN‐oxidoreductase conformational changes of tertiary and secondary structure of enzymes, because red shift was not recorded for the fluorescence spectra of both enzymes. Thus, it is clear that glycerol and sucrose are not aggressive media in relation to the studied proteins. The intensity of maximum emission of proteins reduced with increasing concentrations of glycerol and sucrose, which is connected with the concentration quenching of external tryptophan. In the presence of sucrose has been registered blue shift of the fluorescence spectra of the enzymes. The blue shift of maximum emission of luciferase could be explained by decreasing of solvent effect on the tryptophan residues, exposed to the environment. The blue shift of NAD(P)H:FMN‐oxidoreductase may be due to the mobility decreasing of fluorophores, resulting from the less intensive contact of chromospheres of oxidoreductase with the solvent. In summary, the results presented in this work show that glycerol and sucrose reduce the luciferase and NAD(P)H:FMN‐oxidoreductase inactivation rate and preserve its conformation against thermal unfolding. This work was supported by the Federal agency of science and innovations (contract No 02.740.11.0766), President of RF (grant Leading scientific school No 64987.2010.4), the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058, 11. G34.31.0013). References Sukovataya IE, Tyulkova NA. Kinetic analysis of bacterial bioluminescence in water‐organic media. Luminescence 2001;16:271‐3. Tjulkova NA. Purification of bacterial luciferase from Photobacterium leiognathi with use FPLS‐ system. In Biological Luminescence, Iezowska‐Trzebiatowska B (ed.). Singapore: World Scientific, 1989:369–74. New nanophotonic detection method of carcinogenic polycyclic aromatic hydrocarbons by the example of benzo(a)pyrene Olga A. Sushko, Olena M. Bilash and Mykola M. Rozhitskii Laboratory of Analytical Optochemotronics, Kharkiv National University of Radio Electronics,14 Lenin Ave, 61166, Kharkiv, Ukraine E‐mail: rzh@kture.kharkov.ua Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals formed during the incomplete burning of coal, oil, gas, wood, garbage or other organic substances such as tobacco and charbroiled meat. There are more than 100 different PAHs which are used in medicines and for production of dyes, plastics, pesticides ect. Also PAHs are contained in asphalt used in road construction, in crude oil, coal, coal tar pitch, creosote and roofing tar. PAHs are found throughout in air, water and soil. In air PAHs can form complexes with dust particles while in water, soil, solid sediments PAHs can exist as separate non‐soluble molecules. PAHs can be transformed by photochemical and/or chemical reaction to long‐living product with life‐time from days to weeks (1). Benzo(a)pyrene (BP), C20H12, is a five‐ring PAH which metabolites are mutagenic and highly carcinogenic. This means that BP is a procarcinogen, and the mechanism of BP carcinogenesis depends on its enzymatic metabolism to the ultimate mutagen, benzo(a)pyrene diol epoxide. The last intercalates into DNA, bonding covalently to the nucleophilic guanine bases (2). Therefore the very important problem is the definition of organic polyaromatic carcinogens, the most hazardous among which is BP, in water and other objects by efficient and cheap methods and instruments. There are a number of methods for the PAHs determination in water including high‐performance liquid chromatography, immuno‐chemical analysis, chemical and biological test methods (3). But these methods have several disadvantages, including complexity and high cost of the equipment, sample preparation and analysis procedure, not enough detection limit and selectivity, rather high cost and long assay duration. So the development of novel methods and instruments for the definition of low content of carcinogenic polyaromatic compounds is quite urgent task. Above‐mentioned disadvantages are practically absent in the proposed nanophotonic assay method and sensor device. The nanophotonic method under consideration is based on electrochemiluminescent analysis and modern nanomaterials – quantum‐dimensional semiconductor structures used in the developed nanophotonic sensor's device. The developed sensor itself represents a very small by its dimensions thin layer cell with two or more electrodes intended both for electrochemical and luminescent assays. The working electrode surface inside the sensor's active volume is being modified by Langmuir‐Blodgett or spin‐coating methods with quantum‐dimensions structures such as quantum dots or quantum tubes used as detector elements. The investigation of the developed method and sensor's device show high performance and metrological characteristics such as a low detection limit (< 1 nM), low assay duration and cost, high selectivity and reproducibility. The authors gratefully acknowledge the support for this research by Science and Technology Center in Ukraine Project 5067 (Project Manager: Prof. Rozhitskii M.M). References Moiz Mumtaz, Julia George. Toxicological Profile for Polycyclic Aromatic Hydrocarbons. U.S. Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry. 1995;246–9. Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hotspots in P53. Science. October 18, 1996;274(5286):430–2. Bilash OM, Galaichenko OM, Sushko OA, Rozhitskii M.M. “New nanophotonic detection method of benzo(a)pyrene. Scientific Council on " Analytical Chemistry" . Ukraine. May 2011;58:16–20. Thermal stability of coupled enzyme system NADH:FMN‐oxidoreductase–luciferase in solvents of different viscosity OS Sutormin, IE Sukovataya and VA Kratasyuk Siberian Federal University, Krasnoyarsk, Russia E‐mail: olegsutormin@yahoo.com Development of physico‐chemical basis of bioluminescence assay, extension of the scopes of bioluminescence assay, increase of luciferase activity and selectivity are of great importance now. It was shown, for example, that viscous environment stabilizes coupled enzyme system of luminous bacteria NADH:FMN‐oxidoreductase‐luciferase (1) and the apparent value of the Michaelis constant is enhanced with increasing concentration of organic solvent (2,3). The aim of this work was the investigation of possibility of stabilization and increase of the coupled enzyme system NADH:FMN‐oxidoreductase–luciferase activity in solvents of different viscosity. The thermostability of the bacterial coupled enzyme system in the absence and presence of mentioned osmolytes (sucrose and glycerol) at different temperatures were compared (Figure 1). The activity of the coupled enzyme system in the absence of osmolytes reached maximum at 25 °C whereas in the presence of these osmolytes, maximum activity has shifted to 35 °C. When the temperature increased above the thermal unfolding temperature (above 45 °C), the coupled enzyme system NADH:FMN‐oxidoreductase–luciferase lost almost all of its activity. However, these observations showed that the optimum temperature of the coupled enzyme system increased for about 10 °C in the presence of these osmolytes. In addition, the finding identical values of the remaining activity in the presence of both osmolytes meant that optimum temperatures of the coupled enzyme system essentially depended on the viscosity of the reaction medium. The remaining activity of the coupled enzyme system in the presence of 5.43 M of sucrose is 20% higher than in glycerol at 25–40 °C. It means that the remaining activity depends not only on the viscosity of the reaction medium, but also on physico‐chemical properties of the used osmolytes. Figure 1. Optimum temperature of the coupled enzyme system NADH:FMN‐oxidoreductase–luciferase in the absence (♦) and presence of 40% sucrose ( viscosity 6,16 mPa) (▲) and 50% glycerol (viscosity 6.05 mPa) (■). Arrhenius plots of the first‐order rate constants for the thermal inactivation of the coupled enzyme system NADH:FMN‐oxidoreductase–luciferase showed that activation energy value (Ea) for the coupled enzyme system NADH:FMN‐oxidoreductase–luciferase in the absence of any osmolytes was 16.00 kJ/mol, whereas in the presence of sucrose, it increased to 40.20 kJ/mol and in the presence of glycerol, it reduced to 6.50 kJ/mol. Therefore, the coupled enzyme system in the presence of sucrose is more thermostable than in the presence of glycerol. Thereby, the activation energy value depends not only on the viscosity of the reaction medium, but also on physico‐chemical properties of osmolytes. In summary, the best protector of the coupled enzyme system NADH:FMN‐oxidoreductase–luciferase against thermal stress is sucrose. Acknowledgements This work was supported by the Federal agency of science and innovations (contract No 02.740.11.0766), the Program of the Government of Russian Federation “Measures to Attract Leading Scientists to Russian Educational Institutions” (grant No 11. G34.31.058, 11. G34.31.0013). References Bezrukikh AE, Esimbekova EN, Kratasyuk VA. Thermoinactivation of Coupled Enzyme System of Luminous Bacteria NADH:FMN‐Oxidoreductase‐Luciferase in Gelatin // Journal of Siberian Federal University. Biology 1, 2011;N4:64–74. Sukovataya IE, Tyulkova NA. Kinetic analysis of bacterial bioluminescence in water‐organic media. Luminescence 2001;16:271–3. Sukovataya IE, Kratasyuk VA, Buka NS. Effect of pH of reaction media on kinetic parameters of coupled enzyme system NADH:FMN‐oxidoreductase–luciferase in solvents of increased viscosity // Luminescence 2010;25:188–9. Electrogenerated chemiluminescence of iridium(III) complexes Kalen N. Swanicka, Sébastien Ladouceurb, Eli Zyzman‐Colmanb* and Zhifeng Dinga* aDepartment of Chemistry, The University of Western Ontario, 1151 Richmond Street, London, N6A 5B7, Canada. Fax: 01 519 661 3022; Tel: 01 519 661 2111 x86161 E‐mail: zfding@uwo.ca bDépartement de Chimie, Université de Sherbrooke, 2500 Blvd de l'Université, Sherbrooke, J1K 2R1, Canada Fax: 01 819 821 8017; Tel: 01 819 821 7922 E‐mail: Eli.Zysman‐Colman@USherbrooke.ca Electrogenerated chemiluminescence (ECL) is a powerful analytical technique that generates excited states through electron transfer between radicals in solution and emits light.1 ECL of Ru(bpy)32+ complexes and its derivatives have been studied extensively and have many applications.1–3 Recently, a few iridium(III) complexes have shown to be ECL active in aqueous and organic media.4 Our research group has explored the synthesis of a series of highly luminescent iridium(III) complexes.5 Here we report the ECL mechanisms, efficiencies and spectra of four selected iridium(III) complexes, 1‐4, Fig. 1, in acetonitrile with 0.1 M TBAPF6 as supporting electrolyte. Fig. 2(A) shows a cyclic voltammogram (CV) of complex 2, overlaid with the ECL‐voltage curve. The corresponding ECL spectrum of complex 2 is shown in Fig. 2(B), displaying a maximum wavelength at 576 nm, similar to its photoluminescence (PL) spectrum at 591 nm5. The ECL efficiency for complex 2 is 6.49% relative to (Ru(bpy)3)(PF6)2 taken as 100 % in acetonitrile. More details will be shown in our presentation. Figure 1. Structures of iridium(III) complexes 1‐4. Figure 2. (A) CV of complex 2 overlaid with the ECL‐voltage curve in acetonitrile with 0.1 M TBAPF6 as supporting electrolyte. (B) ECL spectrum of complex 2. References Miao W. Chem. Rev. 2008;108:2506–53. Tokel NE, Bard AJ. J. Am. Chem. Soc. 1972;94:2862–3. McCord P, Bard AJ, Electroanal J. Chem., 1991;318:91–9. Kim JI, Shin I‐S, Kim H, Lee J‐K. J. Am. Chem. Soc. 2005;127:1614–5. LadouceurS, Fortin D, Zysman‐Colman E. Inorg. Chem. 2011;50:11514–26. Use of bioluminescence assay to verify mechanisms of detoxifying effects of humic substances in heavy metal salt solutions AS Tarasovaa, ES Fedorovab and NS Kudryashevaa,b aSiberian Federal University, Svobodniy 79, 660041, Krasnoyarsk, Russia bInstitute of Biophysics SB RAS, Akademgorodok 50, 660036, Krasnoyarsk, Russia Salts of heavy metals are among the most common toxic pollutants. The toxic effects of metals derive from interaction between free metals and cells. The specific biochemical processes and/or cellular and subcellular membranes are responsible for these interactions (1). Humic substances (HS) can serve as natural detoxifying agents in solutions of metallic salts (2). In current study the toxicity of model solutions of heavy metal salts – Pb(NO3)2, CoCl2, CuSO4, Eu(NO3)3, CrCl3, and K3(Fe(CN)6) were assessed by luminous bacteria Photobacterium phosphoreum and bioluminescent system of coupled enzyme reactions NADH:FMN‐oxidoreductase – luciferase. NADH, organic reducer, is a component of a bioluminescent enzyme assay system. The rate of NADH oxidation can serve as an indicator of intensification (or slowdown) of processes in the assay system under the influence of HS in metal salts solutions (3). It was found that HS increased the rates of biochemical processes in the bioassay system with endogenous oxidizer FMN included, but they did not change (or changed slightly) the rates in the presence of the exogenous oxidizers CuSO4 and K3(Fe(CN)6). Hence, the HS made the endogenous processes more competitive in solutions of inorganic toxicants of oxidative nature. Ultrathin sections of the intact bacteria cells were examined with electron microscope. The bacteria were grown in the absence and presence of HS in CrCl3 solution, taken as an example. In the presence of HS, a large number of bacteria were found to contain a polysaccharide layer on the outside of the cell wall. Similar effects were observed in our previous study (2) under addition of HS into solutions of organic oxidizers, quinones. It is known that polysaccharide capsules protect bacteria from antimicrobial agents, and they are almost always present on the surface of cells growing in nature. In our experiments, bacteria could intensify the synthesis of extracellular polysaccharide slime layers under detoxifying action of HS as a response to unfavorable influence of CrCl3. Hence, the mechanisms of detoxification of metal salts solutions by HS were shown to be complex; biochemical and cellular aspects were found to condition those. The detoxifying effects were attributed to: “external” processes of metal‐HS binding in solutions, “internal” changes of biochemical process rate in the bioassay system, and protective response of a cell as a whole. Acknowledgements The work was supported by: the Grant of Ministry of Education and Science RF N 2.2.2.2/5309; Federal Target Program " Research and scientific‐pedagogical personnel of innovation in Russia” for 2009‐2013 years, contract N 02.740.11.0766. References Hodgson EA. textbook of modern toxicology, 3rd edn. Wiley, Canada, 2004. Fedorova E, Kudryasheva N, Kuznetsov A, Mogil'naya O, Stom D. Bioluminescent monitoring of detoxification processes: activity of humic substances in quinone solutions. J Photochem Photobiol B. 2007;88:131–6. Tarasova AS, Stom DI, Kydryasheva NS. Bioluminescent toxicity monitoring of oxidizer solutions: effect of humic substances. Environ Toxicol Chem. 2011;30:1013–7. Structural basis for color modulation mechanism of firefly luciferase bioluminescence Kanako Terakadoa, Ryosuke Yoshimunea, Keiko Gomib, Naoki Kajiyamab, Hideyuki Ikeuchic, Jun Hiratakec, Hiroaki Katoa and Toru Nakatsua aDepartment of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University b Research and Development Division, Kikkoman Corporation c Institute for Chemical Research, Kyoto University Firefly luciferase and its single amino acid substituted mutants emit various ranges from yellow‐green to red (560‐610 nm). We already solved the crystal structures of Japanese firefly luciferases (Luciola cruciata), wild type and red mutant S286N (Ser 286 is replaced with Asn), in complex with an acyl adenylate intermediate analogue in order to investigate the structural basis for the emission color difference in luciferase bioluminescence1. Comparison of these structures showed the movement of the side chain of Ile 288 towards the luciferin moiety only in the wild type. This result suggested that the bioluminescence color might be affected by the difference of the spatial complementarity of excited oxyluciferin and luciferase. In order to confirm our proposed mechanism, we investigated other five mutants with red emission. We focused on the two hydrogen bonding networks between Ser 286 and Tyr 257, and between Arg 220, Asn 231 and oxyluciferin. We solved the X‐ray crystal structures of the five red mutants, Y257F, Y257A, R220A, N231A and N231D in complex with an acyl adenylate intermediate analogue DLASA (5′‐O‐(N‐(dehydroluciferyl)‐sulfamide)adenosine). Bioluminescence spectra of all mutants were red shifted. The emission maxima and the shapes of the spectra of the mutants had various patterns (Figure 1). In the crystal structures of Y257F (λmax = 578 nm) and Y257A (λmax = 608 nm), the movements of Ile 288 such as wild type were not observed (Figure 2). In addition, deletion of the benzene ring by Y257A expanded the cavity of oxyluciferin binding site compared with Y257F. We consider that the difference of spatial complementarity caused the emission difference between Y257F and Y257A. Otherwise, in the crystal structures of R220A (λmax = 595 nm), N231A (λmax = 593 nm) and N231D (λmax = 597 nm), we observed the disruption of hydrogen bonding network between Arg 220, Asn 231 and oxyluciferin, although the movements of Ile 288 were observed as well as wild type. This result suggests that this hydrogen bonding network between Arg 220, Asn 231 and oxyluciferin is essential for yellow‐green emission. Figure 1. Bioluminescence spectra of luciferase wild type and the mutants. Figure 2. Crystal structures of wild type and red mutants complexed with DLASA. Reference 1. Nakatsu N, Ichiyama S, Hirateke J, Saldanha A, Kobashi N, Sakata K and Kato H. Structural basis for the spectral difference in luciferase bioluminescence. Nature 2006;440:372–6. Quantum chemical study of 2‐hydroperoxycoelenterazine generation FN Tomilina,c, LV Tikhonovac, EV Eremeevab,c, SG Ovchinnikova,c and ES Vysotskib,c aInstitute of Physics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia bInstitute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk 660036, Russia cSiberian federal university, Krasnoyarsk 660036, Russia E‐mail: felixnt@gmail.com The Ca2+‐regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. All photoproteins have 2‐hydroperoxycoelenterazine (CLZ‐OOH) substrate bound with hydrogen and Van‐der Waals bonds. Bioluminescence of photoproteins is triggered by binding of three Ca2+ ions. The binding of the ions to the protein induces an oxidative decarboxylation of the CLZ‐OOH with generation of exited protein bound product (coelenteramide). Coelenterazine (CLZ) binding with apo‐protein is a millisecond‐scale process (1). The rate‐limiting step of the active photoprotein formation is the conversion of CLZ to peroxy derivative. According to previously proposed mechanism (2), the first step is deprotonation of CLZ with assistance of a base. However, the crystal structures of obelin and aequorin reveal no amino acid side chains nearby the N7 atom of CLZ, which would function as a base. An alternative mechanism for the formation of 2‐hydroperoxycoelenterazine can be proposed. We reasonably assume that following fast binding coelenterazine undergoes tautomerization from the CLZ N7‐protonated form to CLZ C2‐protonated form. The tautomeric form reacts with oxygen to yield CLZ‐OOH which is stabilized by hydrogen bonding with the hydroxyl group of tyrosine. Ab‐initio Hartree‐Fock and density‐functional calculations were used to study atomic and electronic structure of CLZ isomeric forms and CLZ‐OOH. Semiempirical PM3 and PM6 methods were used to calculate activation energy of CLZ‐OOH formation reaction. The schematic of CLZ‐OOH formation as well as energy reaction profile are presented in Figure 1. The process can be interpreted as one‐stage reaction with proton transferred to oxygen followed by CLZ‐OOH formation. Activation barrier and enthalpy of the reaction are calculated to be 68 kJ/mol and 60 kJ/mol, respectively. We assume that the water molecule and/or Tyr138 redistribute the charges of coelenterazine molecule via polarizing N1‐nitrogen and changing its partial atomic charge. As a result of this inductive effect the C2‐H bond becomes also polarized and the dioxygen molecule becomes situated at 2.4 Å from C2‐protonated carbon under London dispersion force influence. Thus, we believe that formation of a stable van der Waals complex between CLZ C2‐protonated form and oxygen could create a precondition for oxygen to change its spin during the reaction. Our calculations also show that molecular oxygen could spin‐flop from triplet to singlet state during chemical reaction as a result of distance changing between two atoms in the molecule (Figure 1). Activation energy value of 2‐hydroperoxycoelenterazine formation reaction obtained from quantum‐chemical calculations is in a good agreement with experimental kinetic data (Ea = 45.0 kJ/mol, ΔH = 42.6 kJ/mol). Proposed mechanism allows us to describe correctly the formation of CLZ‐OOH without assistance of a base. The mechanism of CLZ‐OOH formation from coelenterazine protonated at C2 carbon is believed to be much more reasonable. Figure 1. Schematic of reaction profile of 2‐hydroperoxycoelenterazine formation from coelenterazine protonated at C2 carbon and O2 molecules in singlet and triplet station. Acknowledgments The work was supported by the Program of the Government of Russian Federation “Measures to attract leading scientists to Russian educational institutions” (grant No 11. G34.31.058), FCP GK‐P333 and grant of SBRAS. We are grateful for the use of computation facilities of Joint Supercomputer Center of the Russian Academy of Sciences MVS‐100K (Moscow) and Center of high‐efficiency calculations of IKIT SFU (Krasnoyarsk). References Eremeeva EV, Markova SV, Westphal AH, Visser AJ, van Berkel WJ, Vysotski ES. FEBS Lett. 2009;583:1939–44. Kondo H, Igarashi T, Maki S, Niwa H, Ikeda H, Hirano T. Tetrahedron Lett. 2005;46:770–7704. Origin and evolution of photogenic tissue in larval fireflies P Tonollia, FC Abdallaa and VR Viviania,b aLaboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring, Universidade Federal de São Carlos (UFSCAR), Sorocaba, Brazil bGraduate Program of Evolutional Genetics and Molecular Biology, São Carlos,SP, Brazil E‐mail: viviani@ufscar.br The origin of beetle bioluminescence remains a mystery. In fireflies, luminescence is found in all life stages, in the eggs, and in lanterns in larval, pupal and adult stages. However, the anatomical origin of the lantern tissue was until recently unknown. Through CCD imaging, histological and biochemical analysis using the Brazilian fireflies Aspisoma lineatum and Cratomorphus sp we found that larvae emit a continuous low level of bioluminescence throughout the entire body during all stages, especially from lateral spots along the body and from the thorax. The fat body, which consists of witish and pinkish lobes spread all over the body, is the source of this weak luminescence. Further inspection showed that the trophocytes are the weakly luminescent cells, showing an ontogenetic link with lantern photocytes. According to spectral and kinetic studies, different luciferase isozymes were found in the lanterns (λmax = 560 nm) and fat body (λ max= 547 nm). Similarly to fireflies in click beetle larvae and railroad worms the fat body is also weakly luminescent, displaying much lower luciferase specific activities and luciferin concentrations than the lanterns. These studies provide a rationale for the widespread location of lanterns in different bioluminescent beetles, indicating that bioluminescence may have arisen as an amorphous luminescence spread throughout the body as a result of accidental byproduct of fat body metabolic activity, evolving through intermediary stages with lateral luminescent spots spread along the body such as in railroadworm and click beetle larvae, and finally reaching current stage of the two developed ventral lanterns in the 8th abdominal segment found in extant larval fireflies. (Financial support: CNPq and FAPESP). Half a century of the contemporary bio‐ and chemiluminescence: a retrospective view on the very beginning Alexey V. Trofimov and Rostislav F. Vasil'ev Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, ul. Kosygina 4, 119334 Moscow, Russia E‐mail: avt_2003@mail.ru, rfv28@mail.ru Keywords: chemiluminescence; bioluminescence The first symposium on bioluminescence and related topics was held half a century ago at the Johns Hopkins University, Baltimore, MD in 1961 (1), while the first symposium on all types of chemiluminescence took place in Durham, NC in 1965 (2). The peculiar historical fact is that the latter meeting was sponsored by the U.S. Advanced Research Projects Agency, Office of Naval Research, U.S. Army Research Office. Both events furnished the logical consequence of a substantial breakthrough in the scientific field referred to the chemical and biological excited‐state generation. Indeed, the preceding years had been commemorated by the observations of a weak light emission accompanying diverse chemical reactions and biochemical processes (3–6), and it has become clear that the “chemical/biological light” constitutes a general and widespread phenomenon rather than rare and exotic happening. For the first (and the last!) time the papers on all types of chemiluminescence were included into the program of the Durham Symposium (1965), whose topics encompassed chemiluminescence in the gaseous phase (10 papers, in particular, by a future Nobel Prize Laureate J.C. Polanyi), experimental methods (first of all, the 68‐pages long paper by J. Lee and H.H. Seliger on the measurements of absolute quantum yields of chemi‐ and bioluminescence is worth mentioning), chemiluminescence in solutions (17 papers), bioluminescence (only 2 papers, however: the one, by J.W. Hastings, Q.H. Gibson and C. Greenwood, on the elucidation of molecular mechanisms of such a phenomenon, and the other one, by G. Cilento, on the generation and transfer of the electronic excitation in biochemical systems). Organization of such a meeting included sending the collection of papers (the Proceedings volume contained 435 pages) to each author by ordinary post (note that neither Internet nor E‐mail were in use these years!) prior to the Symposium. Thus, at the sessions each paper was taken as read and the speakers had merely ten minutes to revise, augment and/or point out the highlights of their research. Discussion followed each paper, for which contributors had the upper limit of five minutes. This historical event has been crowned by publishing the Symposium materials encompassing 27 papers (2), the first “condensed” knowledge on the chemiluminescence phenomenon. References McElroy W, Glass B (Eds). Light and Life. Baltimore: The Johns Hopkins Press, 1961. Wyman GM (Ed). Symposium on Chemiluminescence: Durham, North Carolina. Photochem. Photobiol. 1965;4(6):957–1248. Vladimirov YuA, Litvin FF. Studies of ultra weak luminescence in biological systems. Biofizika 1959;4(5):601–5. Zhuravlev AI, Polivoda AI, Tarusov BN. A weak luminescence from living tissues and organs. Radiobiologiya 1961;1(3):321–3. Vasil'ev RF, Karpukhin ON, Shliapintokh VYa. Chemiluminescence in the reactions of thermal decomposition. Doklady Academii Nauk SSSR 1959;125(1):106–9. Ahnström G, v. Ehrenstein G. Luminescence of aqueous solutions of substances irradiated with ionizing radiation in the solid state. Acta Chem. Scand. 1959;13(4):855–6. Oxidants and antioxidants in the cigarette smoke. Chemiluminescence monitoring Galina F. Fedorova, Valery A. Menshov, Alexey V. Trofimov, Yuri B. Tsaplev and Rostislav F. Vasil'ev Emanuel Institute of Biochemical Physics, RAS, ul. Kosygina 4, 119334 Moscow, Russia. E‐mail: avt_2003@mail.ru Keywords: chemiluminescence; cigarette smoke; free radicals; antioxidants The pathogenesis of numerous smoking‐related diseases is associated with a development of an oxidative stress (1), and the most aggressive oxidative reactants in cigarette smoke are furnished by reactive oxygen and nitrogen species (2). Apart from free radicals, smoke‐borne peroxides (e.g., H2O2) are of contemporary interest (3). However, the experimental data disclose insignificant amount of the primary H2O2 derived immediately from the smoke. Conversely, the secondary H2O2 generated in the organism after smoking is of certain concern. Our H2O2 assay utilizes luminol as the pertinent chemiluminophore and horseradish peroxidase as catalyst, and its sensitivity enables measuring 0.5 ng/ml of H2O2. Since most of phenolic reagents (potential H2O2 sources) reside in the particulate phase of the cigarette smoke, it generates the larger H2O2 amount compared to the gas phase. Since the direct smoke chemiluminescence is of free‐radical nature (4), it may serve as tool to monitor the total free‐radical flux generated by cigarettes, and herein we discuss the dependence of the chemiluminescence characteristics on cigarette type. The radical scavenging by the smoke polyphenols is of prime interest for the assessment of the smoke antioxidant properties, which may be monitored using the peroxy‐radical chemiluminescence (5). The development of the oxidative stress depends not merely on oxidative and antioxidative species, but also on a content of electrophiles in the smoke. For the latter facet, the chemiluminescence approach provides the facile assay based on the Michael addition of electrophiles to model nucleophiles. The pertinent nucleophiles are furnished by thiols. Indeed, their sulfhydryl (SH) groups constitute easily oxidizable functionalities in proteins, which accounts to a major extent for the development of the oxidative stress in vivo. Besides, the nucleophilic SH moieties are prone to reactions with the electrophilic α, β‐unsaturated carbonyls. In vivo, such processes cause alkylation of cellular proteins and trigger pathological developments. Acknowledgments Generous funding by the British American Tobacco Group Research and Development and the Russian Academy of Sciences is gratefully appreciated. References Fearon IM, Faux SP. Oxidative stress and cardiovascular disease: Novel tools give (free) radical insight. J. Molec. Cell. Cardiol. 2009;47(3):372–81. Pryor WA. Cigarette smoke and the role of free radical species in chemical carcinogenicity. Environ. Health Perspect. 1997;105(S4):875–82. Menshov VA, Trofimov AV, Hydrogen peroxide derived from cigarette smoke: “Pardon impossible, to be sent to Siberia?” Mini‐Rev. Org. Chem. 2011;8(4):394–400. Fedorova GF, Menshov VA, Fedorova, Trofimov AV, Tsaplev YuB, Vasil'ev RF. Towards understanding the nature of chemiluminescence derived from cigarette smoke. Luminescence 2010;25(2):130–1. Fedorova GF, Menshov VA, Trofimov AV, Vasil'ev RF. Facile chemiluminescence assay for antioxidative properties of vegetable lipids: fundamentals and illustrative examples. Analyst 2009;134(10):2128–34. Quantitative analysis of bacteriophage plaque expansion by bioluminescence imaging Dann Turner, Jiuai Sun, Shona Nelson, Vyv Salisbury and Darren Reynolds The overlay agar plaque assay is a primary technique used in lytic bacteriophage research. Plaques are defined as localized, visible zones of clearing within an otherwise confluent bacterial lawn and arise after a primary adsorption and productive infection event forming the focal infective centre (Koch, 1964). A period of phage population expansion follows, mediated by multiple rounds of adsorption to, and lysis of, individual bacteria. Release of multiple progeny particles upon bacterial lysis acts to offset diffusion‐mediated decline in phage particle density and population growth continues if sufficient susceptible bacterial are present. Thus, plaque expansion may be envisioned as a travelling wave of infection, moving radially outwards from the focal infective centre and leaving predominantly lysed bacterial cells in its wake. Investigations of plaque enlargement kinetics in vitro have mainly comprised manual measurements, (digital) time lapse photography and fluorescence microscopy (Alvarez et al., 2007; Lee & Yin, 1996). This work presents a novel method using bioluminescent bacterial reporters, allowing for determination of radial expansion velocities of an enlarging plaque and descriptions of plaque morphology. The propagating hosts of phages Felix O1 and SE01, Salmonella enterica serovars Dublin and Enteritidis, respectively, were transformed by electroporation to express the luxCDABE operon of Photorhabdus luminescens. Light emission was correlated with colony counts using log‐fold dilutions of exponential phase cultures immobilized in 100 μL Luria Bertani broth overlay agar (0.6% w/v agarose) in wells of black microtitre plates (Fig. 1). The detection limit for EMCCD imaging was approximately 105 cfu.ml−1, compared to 104 cfu.ml−1 obtained using a microplate reader. Lawns of bioluminescent reporters grown in overlay agar exhibited similar trends of light emission to those from liquid batch culture (Fig. 1). After an initial lag phase, exponential growth was characterized by a rapid increase in light emission. Stationary phase onset was indicated by a deceleration of the rate of increase of bioluminescence, followed by a period of constant then reducing light emission. Figure 1. Bacterial growth and bacteriophage plaque expansion in LB overlay agar. A. Light emission over time for a growing bacterial lawn of S. Enteritidis. Data are the mean of three independent replicates. Error bars denote standard deviation. B. Area equivalent radius for three expanding plaques of bacteriophage SE01. The Siphovirus SE01 produces relatively large plaques (mean diameters 5.69 ± 1.72 mm, n = 10) when titrated upon S. Enteritidis. From images of light emission, the development of plaques by SE01 could be observed within 2 hours. Individual isolated plaques were identified, cropped from the full‐size time‐lapse image stack and analysed using the open‐source ImageJ (Abramoff et al., 2004). To obtain accurate description of the radial velocity of plaque expansion, a routine was developed using MATLAB to detect the edge perimeter and plot as polar co‐ordinates (Fig. 2). Two distinct phases of enlargement were observed. For the first 8 hours radial expansion occurred at a rate of 0.154 ± 0.026 mm h−1. A deceleration period, coinciding with entry of the bacterial lawn into stationery phase, preceded a greatly reduced expansion rate at 0.014 ± 0.006 mm h−1. As virion and host cell mobility, environmental mixing and diffusion are limited compared to in liquid medium, plaque formation in low concentration agar overlays provides a simple and cost‐effective in vitro model for approximation of the infection, expansion and spread of phage populations within semi‐structured environments (Krone & Abedon, 2008). The use of bioluminescent reporters in conjunction with EMCCD imaging could allow temporal and spatial discrimination of the effects of applying phage preparations to biofilms and to bacteria present on food or material surfaces, giving a quantitative measurement to supplement traditional enumeration of plaque and colony forming units. Moreover, extended longitudinal monitoring may allow for the identification of bacterial population recovery post‐exposure to bacteriophage. References Abramoff MD, Magalhaes PJ, Ram SJ. Image Processing with Image J. Biophotonics International 2004;11:36–42. Alvarez LJ, Thomen P, Makushok T, Chatenay D. Propagation of fluorescent viruses in growing plaques. Biotechnology and Bioengineering 2007;96:615–21. Koch AL. The growth of viral plaques during the enlargement phase. Journal of Theoretical Biology 1964;6:413–31. Krone SM, Abedon ST. Modelling phage plaque growth. In: ABEDON, S. T. (ed.) Bacteriophage Ecology: Population Growth Evolution and Impact of Bacterial Viruses. Cambridge University Press. 2008. Lee Y, Yin J. Imaging the propagation of viruses. Biotechnology and Bioengineering 1996;52:438–42. Figure 2. Acquisition and processing of EMCCD images of plaque formation by bacteriophage SE01. A. Montage of time‐lapse series of EMCCD amplified images of bioluminescence at 2 hour intervals. Images have been contrast adjusted and a median filter (radius = 1 pixel) applied. B. Kymograph plotting diameter as a function of time along a single line of pixels perpendicular to the plaque boundary. C. Detection and fitting of the plaque edge and expression in polar coordinates. “Lumtek” bioluminescent test‐systems NN Ugarova, MI Koksharov, GYu Lomakina, VG Frundzhyan and IV Yashin Lomonosov Moscow State University, Moscow, Russia E‐mail: nugarova@gmail.com To apply in practice the results of our fundamental studies in the field of the firefly bioluminescence we organized Lumtek LLC on the basis of MSU Technopark, have developed and started manufacturing the “Lumtek” bioluminescent test‐systems for clinical microbiology and hygiene control of different samples. The principle of operation of the “Lumtek” test‐systems is based on light detection, emitted by the mixture of sample analyzed and ATP‐reagent. To detect the light, portable luminometer “LUM‐1” is used. “Lumtek” test‐systems are effective substitution of a standard microbiology hygiene control and have no analogues in Russia. The composition of “Lumtek” test‐systems: 1) biochemical reagents required to perform the assay (ATP‐reagent and auxiliary reagents); 2) luminometer “LUM‐1”; 3) protocol of assays. ATP‐reagent and luminometer “LUM‐1”, both developed and manufactured by us are the key components of “Lumtek” test‐systems. ATP‐reagent is a biochemical composition on the basis of thermostable mutant Luciola mingrelica firefly luciferase. This luciferase was created by the directed evolution method and contains 8 mutations of amino acid residues and His6‐tag on the C‐end of the enzyme molecule (fig. 1). The optimization of gene expression allowed to produce preparative quantities of the enzyme (up to 300 mg/ml of culture broth) and to purify the enzyme obtained by the metalo‐chelate chromatography. The specific activity of mutant luciferase exceeds the activity of WT luciferase two times. The residual activity of thermo stable mutant luciferase after incubation at 37 °C for 48 h is 80% (1). The “Lumtek” ATP‐reagent surpasses many commercial analogues by the activity and stability. All auxiliary reagents of “Lumtek” test‐systems also are original, have specifications and registration in “Standartinforma” regulations. Figure 1. Structure of the mutant thermostable Luciola mingrelica luciferase. Luminometer “LUM‐1” – portable (weight ~700 g) photon counter designed for registration of weak signals of visible light (fig. 2). This device was certified and registered in the Register of Measuring Tools as the first device in Russia for quantitative measurement of chemi ‐ and bioluminescence. Distinctive features of “LUM‐1”: high sensitivity; stability of indications; low value of a background signal; a wide dynamic range of measurements; low power consumption; fast availability for service (3 minutes); the long period of non‐failure operation (not less than 5500 hours); possibility of on‐site application. Luminometer “LUM‐1” is supplied with USB port and software for data transfer to PC. Figure 2. Luminometer “LUM‐1”. “Lumtek” test‐systems developed were successfully applied in medicine (in‐vitro antibiotic susceptibility assay of clinical samples; bacteriuria rapid screening test; cleanness/sterility control of surfaces, tools and supplies); in food industry and biotechnology. Reference Koksharov M, Ugarova N. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein engineering, design and selection 2011;24:835–44. A genetically encoded fluorescent protein in echinoderms marks the history of neuronal activity Mark A. Verdeciaa,b, Loren L. Loogerb, Luke Lavisb, Johannes Graumannc, Gail Mandela,d and Paul Brehma aVollum Institute, Oregon Health and Science University, Portland OR 972392 bJanelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn VA 201473 cWeil Cornell Medical College, Doha Qatar 241444 dHoward Hughes Medical Institute, Chevy Chase MD 20815 Since the original identification of GFP from jellyfish and corals, the genetically encoded fluorescent proteins have become mainstream indicators for imaging. Functionally homologous candidates exist in more highly evolved bioluminescent invertebrates, including echinoderms. For example, in brittlestars, stimulus‐evoked bioluminescence is transient, lasting seconds, and emanates from specialized cells (photocytes). Prior to light emission, little or no green fluorescence can be observed. However, concurrent with light emission, an intense green, calcium‐dependent fluorescence develops that persists indefinitely. In an effort to identify the gene responsible for this phenomena in brittle stars the chromatographic steps for the purification of the bioluminescent/fluorescent protein were determined. Transcriptomics approaches were used to identify candidate genes which have been isolated and expressed recombinantly. The long term goal is to develop this photoprotein into a genetic marker for long term labeling of calcium activity, which may allow for the mapping of neural circuits. From darkness to brightness: origin and artificial evolution of luciferase activity in Zophobas morio mealworm AMP‐ligase (protoluciferase) VR Viviania,b, RA Pradob and JA Barbosa3 aLaboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate program of Biotechnology and Environmental Monitoring, Federal University of San carlos (UFSCAR), Sorocaba, BrazilbGraduate Program of Evolutional genetics and Molecular Biology, UFSCAR, Brazil E‐mail: viviani@ufscar.brcGraduate Program of Genomics and Biotechnology, catholic University of Brasilia, Brasilia, Brazil Beetle luciferases evolved from AMP‐ligases. However, it is unclear how the new oxigenase/luciferase activity arose and evolved in AMP‐ligases. Several years ago we discovered a luciferase‐like enzyme in Tenebrio molitor mealworms, which was able to produce weak red chemiluminescence in presence of ATP and D‐luciferin. Despite some luciferase homologs having been cloned, the identity of true luciferase‐like enzymes able to produce light remained unknown for long time. We recently found that such luciferase‐like enzyme is located in the Malpighian tubules of mealworms, and finally cloned one of these enzymes from the closely related Zophobas morio giant mealworm. This luciferase‐like enzyme showed to be a short AMP‐ligase (528 residues), however, distantly related to beetle luciferases, suggesting that the potential for bioluminescence in AMP‐ligases could be more ancient than previously though. It displays a weak, but considerable luciferase activity in the red region of the spectrum (613 nm), being the first reasonable model of protoluciferase, useful to investigate the origin of luciferase activity. Its catalytic constant is 2‐3 orders of magnitude lower than that of typical beetle luciferases such as Photinus pyralis firefly, Phrixotrix railroadworm and Pyrearinus termitilluminans click beetle, but 3 orders of magnitude higher than luciferyl‐adenylate spontaneous chemiluminescence. The luminescence reaction is stereoselective for D‐luciferin isomer, suggesting that stereoselectivity is a key feature for development of oxygenase activity. Modeling studies showed that the luciferin binding site of this protoluciferase is smaller and more hydrophobic than that of beetle luciferases, displaying several substitutions of otherwise conserved residues in beetle luciferases. Through site‐directed mutagenesis, we have replaced its luciferin‐binding site residues by those found in beetle luciferases. Although most of the mutations had negative impact on luminescence activity, the substitution I327I increased the luciferase activity, and resulted in a blue‐shifted bioluminescence spectrum, suggesting that substitution of this position may have been critical for the evolution of luciferase activity. Altogether, these results indicate a possible pathway for the structural origin and evolution of luciferase activity in AMP‐CoA ligases. Finally, through genetic engineering we brought the luminescence activity of this enzyme up to visible levels, helping to develop a new luciferase from an AMP‐CoA‐ligase (protoluciferase). (Financial support: Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPq, Brazil). Multicolor brazilian beetle luciferases: structural origin of bioluminescence spectra, biotechnological and environmental applicability VR Viviania,b aLaboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCAR), Sorocaba, Brazil bGraduate Program of Evolutional Genetics and Molecular Biology, São Carlos, SP, Brazil E‐mail: viviani@ufscar.br Among known luciferases, beetle luciferases are the only ones that can produce a wide variety of colors, ranging from green to red. However, most studies have focused mostly on firefly luciferases from the Northern hemisphere, which were extensively studied under the structural and functional points of view, and were used as bioanalytical reagents and reporter genes. During the past 10 years, aiming at understanding the structural origin and evolution of bioluminescence colors, and to increase the range of bioanalytical applications of bioluminescence, we have cloned a large set of new beetle luciferases from Brazilian fauna, which display distinct kinetic and spectral properties useful for bioanalytical applications: (click‐beetles) Pyrearinus termitilluminans larval click beetle luciferase, which emit the most blue‐shifted bioluminescence color (λmax = 534 nm) among beetle luciferases; Fulgeoschlizus bruchi abdominal lantern luciferase (λmax = 540 nm); (Railroadworms) Phrixotrix viviani green emitting luciferase (λmax = 548 nm); Phrixotrix hirtus red emitting luciferase, the only true natural red emitting luciferase (λmax = 623 nm); (Fireflies) Cratomorphus distinctus (λmax = 548 nm); Macrolampis sp2 (λmax = 564/600 nm) and Amydetes fanestratus (λmax = 538 nm) firefly luciferases. These luciferases were expressed, purified, characterized and used as models to investigate the relationship between structure, bioluminescence colors and pH‐sensitivity. Comparative modeling, site‐directed mutagenesis and chimerization studies identified important structural determinants of bioluminescence colors and pH‐sensitivity, such as the loop between residues 223–235, which shields the luciferin binding site, the residues H310, E311, E354 that are involved in pH‐sensitivity, and other stabilizing active site interactions. Luciferin binding site probing with 2,6 TNS and 1,8 ANS showed that the most blue‐shifted luciferases display more hydrophobic luciferin binding site, whereas the red emitting ones display more polar environments. Luciferyl‐adenylate chemiluminescence studies showed that the emission spectrum may differ form the spectrum started with D‐luciferin and ATP, depending on the luciferase, and that the hydrophobic binding pockets of proteins such as BSA may slightly shift the emission spectra toward the blue. Altogether, the results support the influence of the active site conformation and polarity in bioluminescence color modulation. Finally, some of these luciferases turned out to be valuable tools for bioanalytical purposes: Phrixotrix red emitting luciferase is being used in multicolor reporter systems for mammalian cells, and displays applicability as bioanalytical reagent for hemoglobin rich samples; Pyrearinus termitilluminans green emitting click beetle luciferase displays bright signal, slower kinetics and stability valuable for in vivo cell imaging; Macrolampis luciferase displays a very sensitive bimodal spectrum, being useful for intracellular biosensors. These and other new luciferases, such as the blue‐shifted Amydetes and Fulgeoschlizus luciferases, which display high affinity for ATP and shifted optimum pH, are being evaluated for their applicability in cell biosensors for environmental monitoring and bioimaging (Financial support: FAPESP and CNPq, Brazil). The blue‐shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties VR Viviania,b, D Amarala,b, RA Pradoa,b and FGC Arnoldic aLaboratory of Biochemistry and Biotechnology of Bioluminescence, Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), Sorocaba, SP, Brazil; bGraduate Program of Evolutional Genetics and Molecular Biology, São Carlos, SP, Brazil Email: viviani@ufscar.br cRibeirão Preto School of Medicine, São Paulo University, Ribeirão Preto, SP, Brazil Firefly luciferases usually produce bioluminescence in the yellow‐green region, with colors in the green and yellow‐orange extremes of the spectrum being less common. Although several firefly luciferases have been already cloned and sequenced, the three‐dimensional structure solved for two firefly luciferases, and site‐directed mutagenesis studies identified important regions and residues for bioluminescence colors, the structural determinants and mechanisms of bioluminescence colors remain elusive, mainly when comparing luciferases with high degree of divergence. Thus comparison of more closely related luciferases producing colors in the two extremes of the spectrum could be revealing. The South‐American fauna of fireflies remains largely unstudied, with some unique taxa that are not found anywhere else in the world and that produce a wide range of bioluminescence colors, among them fireflies of the genus Amydetes whose taxonomical status as independent subfamily or as a tribe was unclear, and which produce continuous blue‐shifted bioluminescence. Thus, we recently cloned the cDNA for the luciferase of the Atlantic rain forest Amydetes fanestratus firefly, which is found near Sorocaba municipality (São Paulo, Brazil). Despite showing higher degree of identity with the South‐American Cratomorphus, the European Lampyris and the Asiatic Pyrocoelia, phylogenetical analysis of the luciferase sequence placed Amydetes as independent subfamily. This recombinant luciferase displays one of the most blue‐shifted emission spectra (λmax = 538 nm) among beetle luciferases, with remarkable lower pH‐sensitivity and higher affinity for ATP when compared to other firefly luciferases, making this luciferase attractive for sensitive ATP assays. Multialignement and modeling studies showed that the interaction between residues R218, S250 and S347 could be important for stabilizing the luciferin binding site of this luciferase. (Financial support: FAPESP and CNPq). Chemiluminescence of firefly luciferin in deoxygenated DMSO solutions with t‐BuOX (X=Na, K) Rikuo Sibataa, Kouki Hatanakaa and Naohisa Wadab* aFaculty of Science and Engineering, b Faculty of Life Sciences, Toyo University, Gunma 374‐0193, Japan *E‐mail:bhwada@toyo.jp We have focused so far on the spectroscopic properties of firefly lucifering chemiluminescence to find out some intermediates in deoxygenated DMSO with t‐BuOK (1). Here, we investigated the effect of counter‐ions X+ (X = Na, K) on the intermediates produced from the chemiluminescence of firefly luciferin(Ln) in deoxegenated DMSO solutions added t‐BuOX. The Ln intermediates (hereafter called MXs) emit yellow‐green and/or red light without enzyme by pouring oxygen gas into the DMSO solutions. First, Ln intermediates, MNa432 and MK420, produced on adding 5~30 mM of t‐BuOX concentration ((t‐BuOX)), were detected with the absorption peaks at 432 nm and 420 nm, respectively; both intermediates emitted yellow‐green (510nm) and/or red (620 nm) light without enzyme on pouring oxygen gas (Figure 1). Secondly, the intensity of MNa432 chemiluminescence as a whole showed the same tendency as that of MK420 on (t‐BuOX) but the 510 nm‐light intensity of MK420 was reduced largely relative to that of MNa432 in the case of (t‐BuOX) = 30 mM. Moreover, the 510 nm‐light intensity of MK420 ((t‐BuOK) = 30 mM) was increased by dissolving 18‐6 crown ether, K+ clathrate reagent, in the solution. Lastly, the absorption maxima of the products, PNa526 and PK535 formed after the red emissions of MNa432 and MK420, were observed at 526 nm and 535 nm, respectively. The fluorescence maxima of both PNa526 and PK535 were identical with each other at 620nm. On the other hand, the absorption spectra of the products (NNa426 and NK426) formed after yellow‐green light emission, located at the same 426 nm but fluorescence peaks are slightly different at 558 nm and 560 nm, respectively. Figure 1. Integrated chemiluminescence spectra of yellow‐green(a) and red(b) color emissions on pouring oxygen gas in the case of t‐BuONa. According to H‐NMR(data not shown), the intermediate MX generated at 5 ~ 30 mM of (t‐BuOX) is supposed to be a trivalent anion, three protons removed from –OH, ‐COOH and C4H sites of Ln; X+ counter‐ions interact with both of a trivalent MX anion and DMSO solvent molecules and affect the formation pathway of excited oxyluciferin from dioxetanone. Thus we explain qualitatively by our model that: a) the absorption red‐shift (blue‐shift) of MK420 to MNa432 (PK535 to PNa526) and b) the 510 nm‐light intensity of MK420 decreases relatively large compared to that of MNa432 at (t‐BuOX) = 30 mM could be all attributed to the difference of electronegativity and bulk of monovalent ion X+. Reference 1. Shibata S, Yoshida Y, Wada N. Filter‐photometry of chemiluminescence from firefly luciferin intermediate M420 in deoxygenated dimethyl sulfoxide. J. Photoscience 2002;9:290–2. Spectroscopic study on oxyluciferin‐luciferase complex in firefly bioluminescent reaction solution and clues to understand the color tuning mechanism Yu Wanga*, Yuhei Hayamizub and Hidefumi Akiyamaa aInstitute for Solid State Physics, University of Tokyo, Kashiwanoha 5‐1‐5, Kashiwa, Chiba 2778581, Japan bGEMSEC, University of Washington, Seattle, WA 98195, USA *E‐mail: wang@issp.u‐tokyo.ac.jp The color tuning mechanism of firefly bioluminescence still keeps as a controversial issue despite of its wide application. We interpreted that the mere intensity change of green emission determined the spectra of firefly bioluminescence through quantitative study on the absolute spectra at various pH and upon addition of bivalent metal ions (1,2). We observed the conversion from the substrate luciferin (LH2) to oxyluciferin (OL) by monitoring absorption spectra of firefly bioluminescent reaction solution (Figure 1). The stability of OL in luciferase (Luc) and decomposition of their complex were proved by long‐time monitoring of the absorption spectra. The pH dependent absorption spectra of OL‐Luc complex were consistent with those of chemically synthesized OL but showed less sensitivity (3), which was ascribed to the hydrophobic microenvironment provided by the active site of Luc. Figure 1. Time profile of absorption spectra of reaction solution at pH 8.2. The spectra on the left was fitted with the spectrum of LH2 (light grey) and that of OL (dark gray). Fluorescence efficiency of blue and green emission of the OL‐Luc complex at various pH was investigated by selective excitation (Fig. 2). The former showed no dependence on pH and the latter was greatly sensitive to pH. The sensitivity of the fluorescence efficiency of the green emission agreed with the intensity change of green emission in quantitative bioluminescence spectra at various pH, which further supported our proposal. Our results suggested that we should take the emission efficiency of OL into account in addition to its pH equilibrium to explain the color tuning mechanism of firefly bioluminescence. Figure 2. Relative fluorescence efficiency spectra at various pH excited upon 380 nm and 430 nm. References Ando Y, Niwa K, Yamada N, Enomoto T, Irie T, Kubota H, Ohmiya Y, et al. Firefly bioluminescence quantum yield and colour change by pH‐sensitive green emission. Nature Photon. 2007;2(1):44–7. Wang Y, Kubota H, Yamada N, Irie T, Akiyama H. Quantum yields and quantitative spectra of firefly bioluminescence with various bivalent metal ions. Photochem. Photobiol. 2011;87(4):846–52. Naumov P, Ozawa Y, Ohkubo K, Fukuzumi S. Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence. JACS, 2009;131(32):11590–605. Monitoring changes in NF‐kB pathway regulation using highly sensitive multipex bioluminescent reporter assays Brian Webb, Douglas Hughes, Megan Dobbs, Janaki Narahari, Jae Choi and Atul Deshpande Thermo Fisher Scientific, Rockford, IL The study of complex cellular signaling pathways requires powerful and specific tools to monitor changes in gene activation or repression. In order to accurately monitor these processes, reporter gene assays are commonly used. We have developed a series of next generation multiplexed luciferase reporters for studying gene regulation. These reporters were developed to improve the sensitivity and convenience of conventional luciferase reporter systems. First, we have used two naturally secreted luciferase genes, Gaussia luciferase from the Marine copepod Gaussia princeps and Cypridina luciferase from the Marine ostracod Cypridina noctiluca to develop a dual secreted reporter system. This Gaussia/Cypridina dual system enables monitoring transcriptional regulation of two promoters within tissue culture media without the need for cell lysis. Importantly, both Gaussia luciferase and Cypridina luciferase are considerably brighter than traditional Firefly luciferase reporters. Second, we have utilized a mutant form of the Japanese Firefly Luciferase from Luciola cruciata that has a red‐shifted emission spectrum to develop a dual luciferase assay with Gaussia luciferase in which the light output of the two luciferases are spectrally resolvable. This Gaussia/Red Firefly dual spectral assay allows simultaneous monitoring of two promoters in a single read assay through addition of both substrates and then spectral interrogation of the resulting light output. In the present study, we utilized both techniques, multiplexing by spectral separation using Gaussia/Red Firefly, and multiplex assays using the two secretory luciferases, Gaussia/Cypridina, to monitor changes in NFkB promoter activity in response to small molecule agonists. Our results demonstrate the utility of dual secreted luciferase assays for sensitive real time monitoring of NFkB reporter activity in the media and simultaneous detection of spectrally resolvable luciferases using filter based detection. Progress in the preparation of 1,2‐Dioxetanes D. Weiß, D. Ziegenbalg, D. Kralisch and R. Beckert aFriedrich Schiller University, Jena, Institute of Organic Chemistry, Humboldtstr. 10, D‐07743 Jena, Germany bFriedrich Schiller University, Jena, Institute for Technical and Enviromental Chemistry, Lessingstr. 12, D‐07743 Jena, Germany Singlet oxygen is a reagent with many synthetic and practical importances, along a key role in living systems mainly in phenomena like aging and cell signalling (1). It allows very clean and well controlled oxidation and cycloaddition reactions under mild conditions. However, the application of singlet oxygen in synthetic processes is not very popular, because it may depend on the complex methods to generate it. One is the reaction of hypochlorite with hydrogen‐peroxide that allows the formation of singlet oxygen in high concentrations, but due to the reactive starting material and very basic medium it is only useful for particular cases, for example to demonstrate the red emission of singlet oxygen. A better method seems to be the catalytic disproportionation of hydrogen peroxide (2). Starting from olefins this method allows the formation of peroxides also in large scales. In our group we prefer the photochemical generation of singlet oxygen. In this method a solution containing a sensitizer is irradiated with light (3). The sensitizer is excited and transfers his energy to dissolved oxygen, converting the regular triplet oxygen to singlet oxygen. Problems of this method are the solvents and the different solubility of the sensitizer in the given solvent. Further problems are the different absorption maxima of the sensitizers, meaning that every sensitizer needs his own adapted light source. In the history light sources were sodium vapour lamps, halogen lamps or regular bulbs. Beside light these lamps produce a lot of heat and most of the produced light has the wrong wavelength or shines in the wrong direction. Now we use LED as light sources with a small emission angle, less heat production and small but intensive emission bands. This makes it possible to minimize the complexity of the equipment and enables easier and faster handling. In the reactor shown in Fig. 2 it is possible to convert up to one gram of olefin into the corresponding 1,2‐dioxetane or endo‐peroxid; the reaction time depends on the nature of the solvent and the sensitizers employed. The next step is a further miniaturization of the system and a change from a discontinued synthesis in flasks to a continuous synthesis in microtubereactors (4) as well as a change from LED to OLED foils. This work is in progress first results are promising. Figure 2. Our minireactor and his equipment. References Clennan EL, Pace A. Tetrahedron 2005;62:6665–90. Alsters PL, Walther Jary V. Nardello‐Rataj, Jean‐Marie Aubr; Organic Process Research & Development 2010;14:259–62. LFML, Ciscato D, Weiss R, Beckert EL, Bastos FH, Bartoloni WJ. Baader. New Journal of Chemistry 2011;35:773–5. Coyle EE, Oelgemoller M. Photochemical & Photobiological Sciences 2008;7:1313–22. The clinical relevance of certain cytokines, steroid hormones and oxalate in mamma carcinoma patients K Woitkeb, S Albrechtb, W Distlerb and T Zimmermanna aDepartment of Visceral‐ and Vascular Surgery, Hospital Freiberg, Donatsring 20, 09599 Freiberg, Germany bDepartment of Gynecology and Obstetrics, Technical University of Dresden, Fetscherstr. 74, 01307 Dresden, Germany Introduction The macromolecules CA 15‐3 and CEA are established markers for prognosis and progression of breast tumors. Because of the complexity of tumorigenesis it is improbable that a single substance can fulfill such requirements. Possible parameters for the biochemical monitoring are reactive C1‐ and C2‐molecules, that are generated during tumor development (e.g. oxalate). Moreover, IL 6 seems to be associated with the progression of the disease. IL 8, 18, 20 and 21 could also show a correlation with respect to the amount of metastasis. The steroid hormones estrogen, testosterone, androstendione, DHEA and DHEA‐S are known substances with involvement in tumorigenesis. Therefore, the serum concentration during progression of the breast tumor is of particular interest. Figure 1. Hypothetical biochemical pathway by in vivo interactions of oxalate to generate reactive oxygen species. Figure 2. Boxplot with oxalate concentrations of the different subgroups (no tumor/1‐2 sides, p = 0,7; no tumor/3‐4 sides p = 0,033; 1‐2 sides/3‐4 sides p = 0,091). We aim to reveal a possible clinical relevance of the above mentioned biochemical markers in breast cancer patients, taking into consideration the clinical status and the established tumor marker CA 15‐3. Materials and methods The samples of serum were taken from 54 patients of the Kllink für Frauenheilkunde und Geburtshilfe des Universitätsklinikums Carl Gustav Carus. The detection of the tumor marker CA 15‐3 was done using the LIA‐methode of the DiaSorin Company. Oxalate was detected via a chemiluminescence method. The detection of the cytokines was performed with the ELISA method. DHEA, DHEA‐S, androstendione and testosterone were detected with a radioimmunoassay, estrogen with a chemiluninescence immunoassay. Results Oxalate: The tumor free group (mean = 20,88 µmol/l) showed a significant difference compared with the group of 3 to 4 metastasis localizations (mean = 16,24 µmol/l) (mean difference = 4,64 µmol/l / p = 0,033). A little difference between the group with 1 to 2 metastasis localisationes (mean = 20,16 µmol/l) and the group with 3 to 4 metastasis localisations can be recognized (mean difference=3,92 µmol/l / p = 0,091) (t‐Test). IL 6: The tumor free group (mean = 0,13 pg/ml) showed a significant difference compared with the group of 1 to 2 metastasis localisations (mean = 6,97 pg/ml) (p < 0,0005) and the group with 3 to 4 metastasis localisations (mean = 10,00 pg/ml) (p < 0,0005). A statistical difference between the groups of different amounts of metastasis localizations could also be observed (p = 0,087) (Mann‐Whitney‐Test). Conclusions The detected C2 molecule oxalate showed during progression of the disease an inverse correlation compared to CA 15‐3. Independent of inflammatory conditions the serum concentration of IL 6 increased during tumor progression. The analysis of the missing parameters (cytokines and steroid hormones) could reveal, possibly in combination of several biomonitoring molecules (oxalate, glyoxylate, formiate) new perspectives in judging the development of the breast cancer (Fig. 2) The metabolism of oxalate (for hypothetic mechanism see Fig. 1) supports the hypothesis of oxalate playing an interesting pathophysiological role in generating small reactive molecules. Effects of hydrated fullerenes on the luminescence of bacterial luciferase, of whole blood and of bicarbonate water solutions Olga I. Yablonskaya, Vladimir L. Voeikov, Natalia D. Vilenskaya, Svetlana I. Malishenko and Kirill N. Novikov Lomonosov Moscow State University E‐mail: olga.yablonsky@gmail.com It is known that hydrated fullerenes (HyFn) prepared from C60 pristine fullerenes display anti‐oxidant and pro‐oxidant properties due to modification of structural properties of aqueous systems (1). Here we studied the effects of HyFn in the wide range of concentrations including ultra‐low ones (1*10−9 – 1*10−21 M) on the processes accompanied by luminescence taking place in different aqueous systems. It was found that HyFn have a regulatory and protective effect on various types of proteins, including bacterial luciferase of Vibrio fischeri. Surprisingly, HyFn are more effective in ultra‐low concentrations (Figure 1) (3). As protein denaturation occurs mainly because of the damaging by reactive oxygen species, the protective effect of HyFn proves their activity associated with free radicals. Presence of HyFn in the solution also lowered the scattering of the data. Figure 1. Effect of HyFn (10‐13 – 10‐19 M) on heat‐inactivated luciferase. 1—Control non‐heated enzyme; 2 – heat inactivated enzyme; 3 – heat inactivated enzyme with HyFn (10‐19 M) added after heating 4 ‐‐ heat inactivated enzyme with HyFn (10‐13 M) added before heating; 5 ‐‐ heat inactivated enzyme with HyFn (10‐17 M) added before heating;. 6 – heat‐ inactivated enzyme with HyFn (10‐19 M) added before heating. Ordinate – amplitude of the flash of photon emission after the reaction initiation. Each point is the mean ±S.E.M. of 25 measurements. It was demonstrated that addition of HyFn to blood of healthy donors resulted in elevation of lucigenin‐dependent chemiluminescence (CL) and alternation of that in blood of patients with chronic inflamatory diseases. In this case the most pronounced effects were observed with the lowest concentrations used (1*10−6 – 1*10−21 M). It is shown that the pattern of the HyFn influence on the luminescence of blood is individual for each person. Addition of Fe (II) salts (10−5 M) to bicarbonate aqueous solutions induces the development of CL wave, which is amplified by luminol. This indicates that free‐radical processes continuously go on in bicarbonate aqueous solutions. HyFn in low (10−8 M) and ultra‐low (10−22 M) concentrations amplified the intensity of Fe (II)‐induced CL in these solutions (Fig. 2). Thus HyFn are non‐specific modulators of oxygen‐dependent free‐radical processes going on in quite different aqueous systems. Their effects are supposedly related to modification of structural properties of aqueous matrix of all these systems (3). Figure 2. Fe (II)‐induced chemiluminescence of 10 mM bicarbonate aqueous solutions presented as percentage of control samples of bicarbonate solutions without HyFn. References Andrievsky GV, Klochkov VK, Bordyuh A, Dovbeshko GI. Comparative analysis of two aqueous‐colloidal solutions of C60 fullerene 2001. Chemistry Preprint Archive http://preprint.chemweb.com/physchem/0107005. Maltseva EL, Palmina NP, Burlakova EB. Natural (alpha‐tocopherol) and synthetic (phenosan potassium salt) antioxidants regulate the protein kinase C activity in a broad concentration range (10(‐4)‐10(‐20) M). Membr Cell Biol. 1998;12:251–68. Chaplin M. The memory of water; an overview. Homeopathy 2007;96:143–50. Mechanistic insights into chemiluminescent decomposition of firefly dioxetanone Ling Yue and Ya‐Jun Liu* College of Chemistry, Beijing Normal University, Beijing 100875, China E‐mail: yajun.liu@bnu.edu.cn The decomposition mechanism and pathway of the firefly dioxetanone have never been explored at a reliable theoretical level before. The difficulty is that the decomposition process includes biradical, charge‐transfer (CT) and several nearly degenerated states, which is in need of multireference method for considering several states simultaneously with an active space available to the whole reaction process. We have investigated the thermolysis of firefly dioxetanone in both neutral (FDOH) and anionic (FDO−) forms in gas phase and solvents by the multistate second‐order multiconfigurational perturbation (CASPT2) theories in coordination with the ground‐state intrinsic reaction coordinate (IRC) calculated by the Coulomb attenuated exchange‐correlation (CAM‐B3LYP). The size of selected active spaces have been validated adequate large for handing the whole reaction processes of the decompositions of FDOH and FDO−, together with the assistance of state‐average (SA) technique. The decomposition processes of FDOH and FDO– were explored in details. The activation energy of FDO− is 5.9 kcal/mol smaller than that of FDOH by the CASPT2 calculations, which once again indicates the necessary of deprotonation in efficient firefly bioluminescence. According to the present calculations, the chemically initiated electron exchange luminescence (CIEEL) and charge‐transfer initiated luminescence (CTIL) mechanisms were extremely discussed. References Liu YJ, De Vico L, Lindh R. Ab initio investigation on the chemical origin of the firefly bioluminescence, J. Photoch. Photobio. A 2008;194:261–7. De Vico L, Liu YJ, Wisborg Krogh J, Lindh R. The chemiluminescence of 1,2‐dioxetane. Reaction mechanism uncovered, J. Phys Chem. A 2007;111:8013–9. Liu FY, Liu YJ, De Vico L, Lindh R. A Theoretical Study of the Chemiluminescent Decomposition of Dioxetanone, J. Am. Chem. Soc. 2009;131:6181–8. Liu FY, Liu YJ, De Vico L, Lindh R. Towards the Understanding of the Chemically Electron Excitation in Firefly Luminescence: A Theoretical Attempt, Chem. Phys. Lett. 2009;484:69–75. A simple flow injection procedure for the determination of nanogram level norfloxacin in pharmaceutical preparations and biofluids using chemiluminescence detection based on its enhancement of potassium ferricyanide and luminol reaction Li Ying Xi'an Thermal Power Research Institute Co. Ltd., No. 136 Xingqing Road, Xi'an 710032, China E‐mail: liyingmail@163.com Norfloxacin (1‐ethyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(1‐piperazinyl)‐3‐quinolinecarboxylic acid, Figure 1) with molecular weight of 319.24 (C16H18FN3O3) is a fluoroquinolone antibacterial, which exhibits high antimicrobial activity in vitro against a wide variety of gram‐negatives and gram‐positives, including gentamicin‐resistant Pseudomona aeruginosa and methicillin‐resistant Staphylococcus aureus. Norfloxacin has a remarkably broad spectrum of activity and excellent pharmacokinetics allowing for once‐daily dosing. It is widely used to treat human and veterinary diseases and also to prevent diseases in animals. Their main excretion pathway is urinary, and low amounts are found in plasma after a single oral dose of 400–mg norfloxacin. On the other hand, there is concern about the possibility of exposure to low levels of these compounds, resulting in the development of resistance of human pathogens to antibiotics. Figure 1. Structure of norfloxacin. From the literature, different methods were employed for the determination of norfloxacin in pharmaceuticals or biological samples, including high performance liquid chromatography (HPLC) (1), spectrophotometry (2), fluorimetry, mass spectrometry (MS) (3), square‐wave adsorptive voltammetry (4) and atomic absorption spectroscopy (5). Progress in flow‐injection (FI) chemiluminescence (CL) analysis has received much attention in pharmaceutical analysis for its high sensitivity, rapidity and simplicity. The CL for the determination of norfloxacin with different CL systems has been reported, including Ce(IV)‐Na2S2O3, Tb(III)‐nitrate and H2O2‐luminol reactions (6). However, there has been no reported with the luminol‐potassium ferricyanide CL system for the determination of norfloxacin was given so far. It is well know that the reaction between luminol and potassium ferricyanide could emit CL, which has been applied to different fields. In this work, it was observed that norfloxacin could enhance the CL reaction between luminol and potassium ferricyanide. On the basis of this a simple, sensitive and rapid procedure was developed for the indirect determination of norfloxacin. The increment of intensity was linear with norfloxacin concentration over the range from 5.0 ng mL−1 to 1000.0 ng mL−1, with the relative standard deviations (RSD) less than 3.0%, and the detection limit was 1.5 ng mL−1 (3σnoise). The linear regression equation for norfloxacin was ΔICL=9.4063Cnorfloxacin+2.9385,R2=0.9988. At a flow rate of 2.0 mL min−1, a complete determination of norfloxacin, including sampling and washing, could be completed in 0.5 min, offering the sampling efficiency of 120 h−1 accordingly. Interference of foreign species was tested by analyzing a standard solution of norfloxacin into which increasing amounts of interfering analyte was added. Common excipients such as agar and cellulose in capsules caused no obvious interference in the determination of norfloxacin. The proposed procedure was applied successfully to the determination of norfloxacin in pharmaceutical preparations, human urine and serum samples without any pretreatment. The proposed method was practical and suitable not only for quality control analysis but also for complex biological samples, confirming the promise for pharmacological and clinical research. Acknowledgments The author gratefully acknowledges Xi'an Thermal Power Research Institute. References Mansilla AE, Pena AM. HPLC determination of enoxacin, norfloxacin and ofloxacin with photoinduced fluorimetric detection and multiemission scanning application to urine and serum, J. Chromatogr. B 2005;822:185. Rahman N, Ahmad Y, Azmi SN. Kinetic spectrophotometric method for the determination of norfloxacin in pharmaceutical formulations, Eur. J. Pharm. Biopharm. 2004;57:359. Lim J, Park B, Yun H. Sensitive liquid chromatographic–mass spectrometric assay for norfloxacin in poultry tissue, J. Chromatogr. B 2002;772:185. Ghoneim MM, Radi A, Beltagi AM. Determination of norfloxacin by square‐wave adsorptive voltammetry on a glassy carbon electrode, J. Pharm. Biomed. Anal. 2001;25:205. Martnez EJ, Reyes JF, Barrales PO. Terbium‐sensitized luminescence optosensor for the determination of norfloxacin in biological fluids, Anal. Chim. Acta 2005;532:159. Xie ZH, Liao SL, Chen GN. A study on the micelle‐sensitized Ce(IV)‐Na2S2O3‐norfloxacin chemiluminescence system and its applications, Luminescence 2005;20:220. Spectroscopic identification of emitter in electrochemiluminescentreactions with tetraphenylborate anion Yuriy T. Zholudov, Olena M. Bilash, Anatoly V. Kukoba and Mykola M. Rozhitskii Laboratory of Analytical Optochemotronics, Kharkiv National University of Radio Electronics,14 Lenin Ave, 61166, Kharkiv, Ukraine E‐mail: rzh@kture.kharkov.ua Sodium tetraphenylborate (NaTPB) can be used as an analytical reactant in analytical chemistry for determination in aqueous media of certain alkali metal cations such as K+, Cs+, Rb+ and quaternary ammonium compounds due to their precipitation. The chemical properties of TPB are studied quite well whereas only several works are devoted to its electrochemical investigation. First comprehensive electrochemical study of TPB ion oxidation was done by Geske in 1959 (1). It was shown that TPB ion undergoes irreversible two‐electron oxidation at the Pt electrode in acetonitrile. Several later works were aimed at clarification of TPB oxidation mechanism in different solutions including water. At the same time the occurrence of electrogenerated chemiluminescence (ECL) emission from 1,5‐diphenyl‐3‐styrylpyrazoline, 1,5‐diphenyl‐3‐(p‐chlorophenyl)‐pyrazoline and some other luminophors during their oxidation in dimethylformamide solution with NaTPB as a supporting electrolyte, was reported in 1978 (2). The emission was supposed to be due to excited complex of TPB anion and luminophor radical cation decay leading to either direct emission from the exciplex or non‐radiative decay of the later with the luminophor molecules excitation. The purpose of present study is to identify the nature of emitter in ECL reactions with TPB ion and to clarify the mechanism of TPB operation as an ECL coreactant. The spectral studies of ECL emission of different luminophors in dimethylformamide, acetonitrile and aqueous solutions containing TPB ion reveals no evidence of exciplex formation. Fig. 1 shows the ECL spectrum of Ru(bpy)32+ with TPB ion in water as well as its photoluminescence spectrum. Due to a very low ECL intensity for spectral measurements the presented ECL spectrum was averaged from 5 consecutive measurements and smoothed using an adjacent averaging method. Obtained spectrum allows to conclude that in investigated ECL system emission originates from the excited Ru(bpy)32+ species. This fact brings us to a conclusion that excited luminophor state 1A* is formed directly in the homogeneous reaction of its radical cation A+● with products of TPB homogeneous oxidation by A+● i.e. it is the oxidative reduction type of ECL coreactant (3). Figure 1. Photoluminescence (A) spectrum of Ru(bpy)32+ solution in H2O and ECL (B) spectrum of 0.75 × 10−4 M Ru(bpy)3Cl2 · 6H2O+3 × 10−5 M NaTPB in H2O on glassy carbon rotating disk electrode. Supporting electrolyte 0.1 M NaClO4. The proposed mechanism of ECL reaction with TPB coreactant allows further development of analytical systems considering well known property of TPB to precipitate numerous cations in aqueous medium. This work was supported by STCU project #5067 (Project Manager Prof. M. Rozhitskii). References Geske D. The electrooxydation of tetraphenylborate ion; an example of a secondary chemical reaction following the primary electrode process, J. Phys. Chem. 1959;63:1062–79. Rozhitskii N, Bykh A, Kukoba A, Shitov V. Steady‐state electrochemiluminescence in solutions with organometallic electrolytes, J. of Appl. Spectroscopy 1978;28:197–202. Zholudov Y, Bilash O, Kukoba F, Rozhitskii M. Electrogenerated chemiluminescence in systems with tetraphenylborate anion as a coreactant, Analyst, 2011;136:598–604. Figure 1 – Photoluminescence (A) spectrum of Ru(bpy)32+ solution in H2O and ECL (B) spectrum of 0.75×10‐4 M Ru(bpy)3Cl2⋅6H2O + 3×10‐5 M NaTPB in H2O on glassy carbon rotating disk electrode. Supporting electrolyte 0.1M NaClO4. Bed‐side monitoring of ROS and NO in vascular surgery using CL‐Methods T Zimmermanna, M Neubauera and S Albrechtb aDepartment of Visceral‐ and Vascular Surgery, Hospital Freiberg, Donatsring 20, 09599 Freiberg, Germany bDepartment of Gynecology and Obstetrics, Technical University of Dresden, Fetscherstr. 74, 01307 Dresden, Germany In the ischemic/reperfusion phase (clamping‐declamping) during vascular surgery procedures, highly reactive oxygen species (ROS) and NO are released in in the blood. NO reacts very slightly with superoxide anions giving rise to the formation of peroxynitrite. The aim of therapeutic approaches must be to counter this fatal reaction mechanism, while inhibiting the reaction of NO with ROS to prevent formation of peroxynitrite. An important fact is that the reperfusion phase in vascular surgery does not end once surgery is finished. We conducted a study with 20 patients suffering from infrarenal aortic aneurysms and 20 patients with stage IIb‐IV peripheral arterial occlusive disease. Blood samples were taken at the following time points (preoperatively, pre‐ and postclamping, at the end of surgery, on postoperative days 1, 2, 3 and 7. ROS and NO were measured op‐side and bed‐side using chemiluminometric methods. Superoxide anion and hydrogen peroxide generation showed a biphasic course. Concomitantly, persistence of ROS formation was noted up to day 7. NO, too, had a biphasic course.If one studies the patients individually and takes a cut‐off point of 5000RLU/s (measure of NO concentration in whole blood), patients can be divided into those with high NO formation and those with low NO formation. The reverse trend was observed for superoxide anion concentrations in the blood, both in the case of POAC and aortic aneurysm patients. This is suggestive of a dynamic equilibrium between NO and superoxide anions, and is to be interpreted as an indirect sign of peroxynitrite formation. Therapeutic approaches must now be targeted at this! To prevent the positive effects of NO and the negative effects of peroxynitrite inhibition of ROS generation or inactivation of ROS appears to be a promising therapeutic option. Sodium selenite can be used as a therapeutic agent.. To verify whether selenite is really an efficient superoxide anion scavenger, we conducted real‐time, intraoperative determination of ROS and NO at the bed‐side and op‐side during vascular surgery operations. Thanks to this immediate determination of NO and superoxide anions, we were able to control selenite treatment already intraoperatively, while ensuring that NO was not converted into peroxynitrite but, rather, was preserved at a high concentration. If there was an increase in superoxide anions, we administered selenite, providing for timely control of a successful outcome. The patient who had received selenite showed lower superoxide anion concentrations and high NO concentration, something we interpreted as being an indirect sign of inhibited peroxynitrite What requirements should be addressed to therapy for the ischemic/reperfusion damage occurring during vascular surgery procedures Start treatment in the early reperfusion phase Bed‐side and op‐side determination of ROS and NO is necessary (real‐time) ‐ using CL‐methods Immediate treatment with antioxidant Immediate treatment control through bed‐side and op‐side determination of ROS and NO‐ CL‐methods Treatment optimization Copyright © 2012 John Wiley & Sons, Ltd.
Journal
Luminescence: the Journal of Biological and Chemical Luminescence – Wiley
Published: Mar 1, 2012