Spectrochimica Acta Part A 54 (1998) 175–183
Correlation between dynamics, structure and spectral properties
of human h1-acid glycoprotein (orosomucoid): a fluorescence
approach
J.R. Albani *
Laboratoire de Biophysique Mole´culaire, Uni6ersite´ des Sciences et Technologies de Lille, BP
649
,
59656
Villeneu6ed
’
Ascq Ce´dex, France
Received 9 May 1997; received in revised form 9 September 1997; accepted 11 September 1997
Abstract
Dynamics of proteins and membranes are usually investigated by red-edge excitation spectra and fluorescence
anisotropy. In a viscous or rigid medium, the fluorescence maximum position changes with the excitation wavelength
upon red-edge excitation. In addition to the shift in the emission maximum on red edge excitation, fluorescence
anisotropy is also known to be dependent on the excitation and emission wavelengths in viscous media. However, this
dependence has always been explained by the fact that the fluorophore is rigid, i.e. it does not diplay any residual
motions. The aim of the present work was to check the validity of this latest assumption and to explain the possible
origin of the dependence of the anisotropy on both the excitation and emission wavelengths. Therefore, we compared
the results obtained from the fluorescence of the Trp residues of two h1-acid glycoproteins (orosomucoid). One
protein was purified by chromatographic methods (orosomucoid
c
) and the other was obtained with ammonium
sulfate precipitation (orosomucoid
s
). Trp residues of orosomucoid
c
display free motions while those of orosomucoid
s
are rigid. The general qualitative feature of the excitation anisotropy spectra recorded on both types of preparation
is identical and resembles that obtained for other proteins containing tryptophan residue in protein. The fluorescence
anisotropy measured across the emission spectra decreases for both preparations, indicating that this phenomenon is
characteristic for fluorophores surrounded by a rigid microenvironment or by a microenvironment that displays
motions. The fluorescence anisotropy variation across the emission and the excitation spectra is more important when
the fluorophore possesses constrained motions than when it displays a high degree of freedom. Our results clearly
demonstrate that the tertiary structure of the protein and the structure and dynamics of the microenvironments of the
Trp residues are the origin of the dependence of anisotropy on the excitation and emission wavelengths. © 1998
Elsevier Science B.V.
Keywords
:
h1-acid glycoprotein (orosomucoid); Protein dynamics and structure; Red-edge excitation spectra; Fluores-
cence anisotropy
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