FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λm), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield (q) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λm 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λm# 350–353 nm, ΔΛ= 59–61 nm, q# 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λm# 340–342 nm, ΔΛ= 53–55 nm, q# 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.

[1]  F Noack,et al.  Investigation of protein hydration by proton spin relaxation time measurements. , 1970, Biochimica et biophysica acta.

[2]  J. Feitelson.  THE FORMATION OF HYDRATED ELECTRONS FROM THE EXCITED STATE OF INDOLE DERIVATIVES , 1971 .

[3]  R. Steiner,et al.  Excited states of proteins and nucleic acids , 1972 .

[4]  J. Longworth.  EXCITED STATE INTERACCIÓNS IN MACROMOLECULES * , 1968 .

[5]  H. Schwan.  ELECTRICAL PROPERTIES OF BOUND WATER , 1965 .

[6]  P. Wahl,et al.  Effect of solvent upon the fluorescence decay of indole. , 1971, Biochimica et biophysica acta.

[7]  R. Steiner,et al.  The tryptophan microenvironments in apomyoglobin. , 1970, The Journal of biological chemistry.

[8]  Arthur W. Luehrmann.  Proceedings of the Second Annual Conference on Computers in the Undergraduate Curricula (Dartmouth College, Hanover, New Hampshire, June 23-25, 1971). , 1971 .

[9]  A. Glazer,et al.  Spectroscopic studies on papain and some inactive derivatives. , 1969, The Journal of biological chemistry.

[10]  M. Walker,et al.  Exciplex Studies. II. Indole and Indole Derivatives , 1967 .

[11]  F. Teale,et al.  Resonance energy transfer in pepsin conjugates. , 1969, Journal of molecular biology.

[12]  J. Eisinger,et al.  Fluorescence quenching and isotope effect of tryptophan. , 1969, The Journal of chemical physics.

[13]  N. Sharon,et al.  A spectrofluorometric study of tryptophan 108 in hen egg‐white lysozyme , 1970, FEBS letters.

[14]  L. Sluyterman,et al.  An agarose mercurial column for the separation of mercaptopapain and nonmercaptopapain. , 1970, Biochimica et biophysica acta.

[15]  R. Lumry,et al.  EXCIPLEX STUDIES–V. ELECTRON EJECTION FROM INDOLE AND METHYL‐INDOLE DERIVATIVES * , 1972 .

[16]  S. Lehrer.  The selective quenching of tryptophan fluorescence in proteins by iodide ion: lysozyme in the presence and absence of substrate. , 1967, Biochemical and biophysical research communications.

[17]  I. Z. Steinberg.  Long-range nonradiative transfer of electronic excitation energy in proteins and polypeptides. , 1971, Annual review of biochemistry.

[18]  M. Brunori,et al.  Fluorescence studies of Aplysia and sperm whale apomyoglobins. , 1970, Biochemistry.

[19]  R. Steiner,et al.  Luminescence of the tryptophan and tyrosine residues of papain in solution. , 1970, Biochemistry.

[20]  H P Schwan,et al.  Dielectric dispersion of crystalline powders of amino acids, peptides, and proteins. , 1965, The Journal of physical chemistry.

[21]  F. Teale,et al.  The ultraviolet fluorescence of proteins in neutral solution. , 1960, The Biochemical journal.

[22]  Raymond F. Chen,et al.  Fluorescence Decay Times: Proteins, Coenzymes, and Other Compounds in Water , 1967, Science.

[23]  A. Finazzi-Agro’,et al.  Environment of copper in Pseudomonas fluorescens azurin: fluorometric approach. , 1970, Biochemistry.

[24]  B. Jennings,et al.  Discussion on the shape of bovine plasma albumin. , 1966, Biochimica et biophysica acta.

[25]  B. Schoenborn.  A neutron diffraction analysis of myoglobin. 3. Hydrogen-deuterium bonding in side chains. , 1972, Cold Spring Harbor symposia on quantitative biology.

[26]  P. Wahl,et al.  Fluorescence studies on human serum albumin. , 1971, Biochemical and biophysical research communications.

[27]  Burshteĭn Ea.  [Quenching of protein fluorescence. I. Principles of the method. Solutions of tryptophan, tyrosine, and denaturated proteins]. , 1968 .

[28]  R. Cowgill.  Fluorescence and protein structure. XVII. On the mechanism of peptide quenching. , 1970, Biochimica et biophysica acta.

[29]  G. Weber,et al.  Failure of Energy Transfer between Identical Aromatic Molecules on Excitation at the Long Wave Edge of the Absorption Spectrum. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Walker,et al.  EXCIPLEX STUDIES‐IV. RADIATIVE AND NON‐RADIATIVE RELAXATION OF THE FLUORESCENT STATE OF INDOLE † , 1971 .

[31]  S. Lehrer.  Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. , 1971, Biochemistry.

[32]  G. Weber,et al.  Determination of the absolute quantum yield of fluorescent solutions , 1957 .

[33]  R. Steiner.  Varying luminescence behavior of the different tryptophan residues of papain. , 1971, Biochemistry.

[34]  Y. Yamamoto,et al.  Spectroscopic studies on the configurational structures of ribonuclease T 1. , 1970, Biochimica et biophysica acta.

[35]  M. Shinttzky,et al.  Fluorometric Detection of Histidine‐Tryptophan Complexes in Peptides and Proteins , 1967 .

[36]  M. Kronman,et al.  THE FLUORESCENCE OF NATIVE, DENATURED AND REDUCED‐DENATURED PROTEINS * , 1971 .