Chemical Biology & Drug Design

PRAKASH, V.; NANDI, R. K.; JIRGENSONS, B.

doi: 10.1111/j.1399-3011.1980.tb02906.xpmid: 6158490

The circular dichroic spectra of α‐globulin have been obtained under various solution conditions of sodium dodecyl sulfate, acid, alkali, urea and guanidine hydrochloride. The protein in phosphate buffer pH 7.4, 0.2 M has about 25%β‐structure and 5%α‐helix, the rest being aperiodic or irregular structure. Sodium dodecyl sulfate induced more α‐helical structure in the protein. The protein had nearly 20%α‐helix at 1 times 10‐2 M SDS. At extreme acid or alkaline pH, the protein had no α‐helix with β‐structure decreasing with further extremes of pH. The protein is represented by 100% aperiodic structure in 6.6 M urea and in 6.0 M guanidine hydrochloride solutions. The above results are discussed in view of some of the earlier results with regard to the association‐dissociation and denaturation behavior of α‐globulin under various solution conditions.

BRESLOW, ESTHER; STAHL, GLENN L.; WALTER, RODERICH

doi: 10.1111/j.1399-3011.1980.tb02907.xpmid: N/A

The role of the α‐amino group of oxytocin in affecting the conformation of oxytocin and its binding to neurophysin was studied by a comparison of the circular dichroism and binding properties of oxytocin with those of (L‐2‐hydroxy‐3‐mercaptopropionic acid)oxytocin which contains a hydroxyl in place of the oxytocin amino group. The circular dichroism properties of (L‐2‐hydroxy‐3‐mercaptopropionic acid)oxytocin were very similar to those of deamino‐oxytocin (in which the amino group of oxytocin is replaced by a hydrogen) but differed significantly from those of oxytocin, particularly under conditions in which the oxytocin α‐amino group is protonated. The protonated oxytocin α‐amino group is known to participate in a salt‐bridge with a neurophysin carboxyl at neutral pH, but oxytocin appears to bind to neurophysin without salt‐bridge formation below pH 2. Nonetheless, (L‐2‐hydroxy‐3‐mercaptopropionic acid)oxytocin, like deamino‐oxytocin, was found not to bind to the hormone‐binding site of neurophysin with measurable affinity at either neutral pH or low pH. The results indicate that, in the binding of oxytocin to neurophysin, the protonated oxytocin α‐amino group plays a role more complex than that of carboxylate charge neutralization and suggest that this role involves an effect on oxytocin conformation. However, highly specific bonding interactions between the α‐amino group and neurophysin, additional to those of salt‐bridge formation, are not precluded.

FAHRENHOLZ, FALK; THIERAUCH, KARL‐HEINZ

doi: 10.1111/j.1399-3011.1980.tb02908.xpmid: 7419359

For the synthesis of p‐azidophenylalanine peptides, the p‐amino group of p‐amino‐L‐phenylalanine is protected with the Z‐ or Boc residue via the copper complex or by specific acylation at pH 4.6. The α‐amino or α‐carboxy group is blocked by a protecting group (Boc, Ddz, OMe respectively Z, Nps, Ddz), which can be removed selectively. The synthesis of nine derivatives of p‐amino‐L‐phenylalanine for incorporation into the peptide chain is described. The p‐amino‐phenylalanine is converted to p‐azidophenylalanine without affecting disulfide bridges.

STAHL, GLENN L.; SMITH, CLARK W.; WALTER, RODERICH

doi: 10.1111/j.1399-3011.1980.tb02909.xpmid: 7419360

Three beaded poly‐N‐acrylyldialkylamine resins have been systematically compared with polystyrene‐co‐1%‐divinylbenzene for their ability to interact with solvents that are useful in peptide synthesis. The three resins have the same cross‐linking monomer, N,N1‐bisacrylyl‐1,2‐diminoethane, and functionalizing monomer, N‐acrylyl‐1,6‐diaminohexane hydrochloride. They differ from one another in the compositions of the base monomer, degree of cross‐linking (DC), quantity of functionalizing monomer, or monomer dilution ratio (MDR, ratio of inert solvent to monomers by weight). Resin1is poly‐N‐acrylylpyrrolidine containing 0.75 mmol/g of amino function with DC 4.4 and MDR 4; resin2is poly‐N‐acrylyldimethylamine containing 0.75 mmol/g of amino function with DC 4.4 and MDR 4; resin3is poly‐N‐acrylyldimethylamine with DC 10 and MDR 6.8. Swelling was the property used as the measure of solvation in a given solvent for the macroscopically insoluble polymers. In their protonated or acylated forms resins 1—3 exhibit favorable swelling properties in methanol, ethanol, 2,2,2‐trifluoroethanol, 2‐propanol, acetic acid, and water, solvents in which polystyrene either swells little or not at all. In addition, the resins swell in methylene chloride, chloroform and dimethylformamide, solvents commonly used in the Merrifield method of solid‐phase peptide synthesis, to about the same extent as polystyrene. The favorable swelling properties of the poly‐N‐acrylyldialkylamine resins should not only allow the use of most of the techniques developed for the solid‐phase method, but should allow the invention and application of new techniques that rely on aqueous or other polar solvents.

GOTO, KASHIKO; TAKAHASHI, NORIKO; MURACHI, TAKASHI

doi: 10.1111/j.1399-3011.1980.tb02910.xpmid: N/A

Stem bromelain was cleaved with cyanogen bromide, and the products were fractionated with and without prior maleylation and sulfitolysis. The fragments that corresponded to the carboxyl‐terminal half of the molecule were isolated and nearly completely sequenced. This portion of the enzyme molecule contained one disulfide linkage. A specific cleavage at the amino peptide bonds of that cystine residue by reduction, modification into S‐cyano derivatives and exposure to alkali gave important information of the amino terminal sequence. By combining the present data with the previously known partial sequence of the parent molecule, 101 amino acid residues were aligned down to the carboxyl terminus and compared with those of papain. The sequence homology between carboxyl‐terminal halves of these two thiol proteases of plant origin was found to be 34.7%.

FELIX, ARTHUR M.; JIMENEZ, MANUEL H.; WANG, CHING‐TSO; MEIENHOFER, JOHANNES

doi: 10.1111/j.1399-3011.1980.tb02911.xpmid: 6106637

Experimental details for practical syntheses of somatostatin and D‐Trp8‐somatostatin are described. The peptides were assembled from three fragments which permit further syntheses of analogs with modifications at positions 1, 2 or 8. Nα‐Bpoc protecting groups were used for the two major fragments and these were selectively removed in the presence of the tert‐butyl derived amino acid side chain functionalities. The two cysteine residues were protected by acetamidomethyl groups. All the peptide intermediates were fully characterized and a 10‐g synthesis of the protected tetradecapeptide is reported. Major fragments were coupled by the azide method in good yield. Dihydrosomatostatin and D‐Trp8‐dihydrosomatostatin were isolated, purified, characterized and cyclized. Polymeric side‐product was successfully recycled (by reduction with dithiothreitol and reoxidation) to give an overall yield for the oxidation of 52%. Somatostatin and D‐Trp8‐somatostatin were purified by gel filtration or countercurrent distribution and the final products were fully characterized and determined to be>97% pure by reversed phase high performance liquid chromatography.

HAN, SHAW‐LIN; RIVIER, JEAN E.; SCHERAGA, HAROLD A.

doi: 10.1111/j.1399-3011.1980.tb02912.xpmid: 6106638

Laser Raman spectroscopy has been used to study the conformations of somatostatin and some selected analogues in aqueous solution. The results indicate that the CS‐SC dihedral angles of somatostatin and of these analogues (except [Ala3,14]‐SS, which has no disulfide bond) are within 20° of ± 85°, and the SS‐CC dihedral angles are predominantly in the range of 50° ‐ 180°. Furthermore, from the behavior of the amide I' and amide III bands, it appears that somatostatin adopts a β‐pleated sheet structure, whereas its analogues are less ordered (to varying degrees).

MOOBERRY, ED. S.; DALLAS, JERRY L.; SAKAI, TED T.; GLICKSON, JERRY D.

doi: 10.1111/j.1399-3011.1980.tb02913.xpmid: 6158491

The acid‐base titration of bleomycin‐A2 in D2O solution at 35 ± 5° has been monitored by 13C n.m.r. spectroscopy at 67.89 MHz. The following pKDa values were obtained: 3.68 ± 0.05 (secondary amine), 5.29 ± 0.03 (imidazole), and 8.23 ± 0.19 (primary amine), where KDa is the dissociation constant in D2O solution. The equilibrium isotope effects (pKDa ‐pKa in H2O) are: 0.70 ± 0.06 (secondary amine), 0.28 ± 0.04 (imidazole), and 0.85 ± 0.19 (primary amine). Titration of the imidazole group of Bleo‐A2 occurs at Nπ, i.e. only Nπ is protonated in basic solution. Significant protonation shifts are almost completely limited to carbons of the N‐terminal tetrapeptide, suggesting that the C‐terminal tripeptide extends into the solvent and interacts to a minimal extent with the rest of the molecule. Long range protonation shifts associated with titration of the imidazole and secondary amine groups indicate that protonation of one or both of these sites is probably accompanied by significant conformational changes. The observed protonation shifts generally fail to correlate with Zn(II) complexation shifts reported by Dabrowiak et al. (1973, Biochemistry 17, 4090) indicating that ligation sites cannot unambiguously be determined from these complexation shifts. The complexation shifts previously attributed to coordination of the imidazole and carbamoyl groups probably result from conformational changes.

TZOUGRAKI, CHRYSA; MAKOFSKE, RAYMOND C.; GABRIEL, THOMAS F.; MICHALEWSKY, JOSEPH; MEIENHOFER, JOHANNES; LI, CHOH HAO

doi: 10.1111/j.1399-3011.1980.tb02914.xpmid: 6252107

A solution synthesis of human β‐endorphin (β‐EP) was carried out by condensation of protected peptide segments bearing Nα‐tert.‐butyloxycarbonyl groups and benzyl‐derived groups for the protection of functionalities in amino acid side chains. Five intermediate segments were assembled in a stepwise manner starting at the carboxyl terminus. Thus, the segment of sequence region (27–31) was coupled to segment (22–26) by the azide method. Segment (19–21) was incorporated into the growing chain by azide coupling, and segment (10–18) by dicyclohexylcarbodiimide coupling in the presence of 1‐hydroxybenzotriazole (DDC‐HOBt). Solubility problems in condensing the ensuing 22‐peptide with segment (1–9) by DDC‐HOBt were overcome by using a dimethylformamidephenol mixture as a solvent. Protecting group cleavage by Na in liquid NH3 was much superior to liquid HF which gave rise to many decomposition products. Homogeneous βn‐EP indistinguishable from authentic material in physiochemical and biological properties, was obtained in a single preparative reversed phase liquid chromatographic step after protecting group cleavage.