Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching.

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.

[1]  D. O'connor,et al.  Time-Correlated Single Photon Counting , 1984 .

[2]  A. Szabo,et al.  Probing alpha-helical secondary structure at a specific site in model peptides via restriction of tryptophan side-chain rotamer conformation. , 1994, Biophysical journal.

[3]  L. Brand,et al.  Time-resolved fluorescence and anisotropy decay of the tryptophan in adrenocorticotropin-(1-24). , 1981, Biochemistry.

[4]  I Munro,et al.  Subnanosecond motions of tryptophan residues in proteins. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[6]  A. Szabo,et al.  Fluorescence decay of tryptophan conformers in aqueous solution , 1980 .

[7]  J J Volwerk,et al.  Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. , 1985, Biochemistry.

[8]  M. Karplus,et al.  Fluorescence depolarization of tryptophan residues in proteins: a molecular dynamics study. , 1983, Biochemistry.

[9]  R. F. Chen,et al.  Removal of fatty acids from serum albumin by charcoal treatment. , 1967, The Journal of biological chemistry.

[10]  M. Ameloot,et al.  Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. Experimental verification on unilamellar vesicles and lipid/alpha-lactalbumin complexes. , 1984, Biophysical journal.

[11]  T. Hennecke,et al.  Long-chain fatty acid-binding to albumin: re-evaluation with directly measured concentrations. , 1994, Biochimica et biophysica acta.

[12]  W. Scheider.  Ligand-independent activated state of serum albumin for fatty acid binding , 1980 .

[13]  E. Gratton,et al.  Rotations of tryptophan residues in proteins. , 1986, Biochemical Society transactions.

[14]  D C Carter,et al.  Structure of serum albumin. , 1994, Advances in protein chemistry.

[15]  N. Heegaard,et al.  Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: New screening techniques for determination of ligand binding to albumin , 1995, Electrophoresis.

[16]  R. Hochstrasser,et al.  Picosecond fluorescence decay of tryptophans in myoglobin. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Janatova,et al.  The heterogeneity of bovine albumin with respect to sulfhydryl and dimer content. , 1968, The Journal of biological chemistry.

[18]  A. J. Bain,et al.  Complete determination of the state multipoles of rotationally resolved polarized fluorescence using a single experimental geometry , 1984 .

[19]  A. Szabo,et al.  CONFORMATIONAL HETEROGENEITY OF TRYPTOPHAN IN A PROTEIN CRYSTAL , 1995 .

[20]  P. Brick,et al.  Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites , 1998, Nature Structural Biology.

[21]  C A Ghiron,et al.  Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. , 1976, Biochemistry.

[22]  F. Soetewey,et al.  Size and shape determination of native and defatted bovine serum albumin monomers. II. Influence of the fatty acid content on the conformation of bovine serum albumin monomers. , 1972, Journal of biochemistry.

[23]  J. Brochon,et al.  Time-resolved fluorescence of the single tryptophan residue in rat alpha-fetoprotein and rat serum albumin: analysis by the maximum-entropy method. , 1990, Biochemistry.

[24]  J. Lakowicz,et al.  Tryptophan fluorescence intensity and anisotropy decays of human serum albumin resulting from one-photon and two-photon excitation. , 1992, Biophysical chemistry.

[25]  B. Valeur,et al.  RESOLUTION OF THE FLUORESCENCE EXCITATION SPECTRUM OF INDOLE INTO THE 1La AND 1Lb EXCITATION BANDS * , 1977, Photochemistry and photobiology.

[26]  P. Callis,et al.  1La and 1Lb transitions of tryptophan: applications of theory and experimental observations to fluorescence of proteins. , 1997, Methods in enzymology.

[27]  T. Imoto,et al.  Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues. , 1998, Biochemistry.

[28]  J. Lakowicz,et al.  Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. , 1973, Biochemistry.

[29]  J. Gollan,et al.  Receptor for albumin on the liver cell surface may mediate uptake of fatty acids and other albumin-bound substances. , 1981, Science.

[30]  H. Nakano,et al.  Formation and Reaction of Carbonyl Ylides. Reactions of 1-Methoxy-2-benzopyrylium-4-olate with Isothiocyanates. , 1992 .

[31]  C. J. Schmidt,et al.  Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase. , 1981, Biochemistry.

[32]  G. Fleming,et al.  Subpicosecond Fluorescence Depolarization Studies of Tryptophan and Tryptophanyl Residues of Proteins , 1992 .

[33]  A. J. Bain,et al.  Strong molecular alignment in anisotropic fluid media , 1996 .

[34]  P. O'shea,et al.  Interactions of macromolecules with the mammalian cell surface. , 1995, Journal of cell science.

[35]  R. Hochstrasser,et al.  Fluorescence and energy transfer of tryptophans in Aplysia myoglobin. , 1987, Biophysical journal.

[36]  M. Otagiri,et al.  Probing the cysteine 34 residue in human serum albumin using fluorescence techniques. , 1997, Biochimica et biophysica acta.

[37]  M. Eftink,et al.  Exposure of tryptophanyl residues and protein dynamics. , 1977, Biochemistry.

[38]  J. Tanaka,et al.  Polarized Absorption Spectra of Crystals of Indole and Its Related Compounds , 1972 .

[39]  K. Kinosita,et al.  A theory of fluorescence polarization decay in membranes. , 1977, Biophysical journal.