Abstract
With the aim of finding non-equilibrium dipole-relaxational electronic excited states of tryptophan residues in proteins the dependence of the fluorescence emission maximum on excitation wavelength was studied for several proteins containing a single tryptophan residue per molecule. Spectral shifts upon red-edge excitation are not observed for short wavelength-emitting proteins (azurin, two-calcium form of whiting parvalbumin, ribonucleases C 2 and T 1). This may be because of the non-polar environment of the tryptophan residues in these proteins or because of the absence of dipole-orientational broadening of spectra. The effect was also not found for proteins emitting at long wavelengths (max. at 341–350 nm) —melittin at low ionic strength, IT-Aj1 protease inhibitor, myelin basic protein. In these proteins, the tryptophan residues are exposed to the rapidly relaxing aqueous solvent. Spectral shifts associated with red-edge excitation are observed for proteins emitting in the medium spectral range — human serum albumin in the N and F forms, IT-Aj1 protease inhibitor at pH 2.9, melittin at high ionic strength as well as the albumin-dodecylsulfate complex. This suggests the existence in these proteins of a distribution of microstates for tryptophan environment with various orientation of dipoles and of slow (on the nanosecond time scale) mobility of the field of these dipoles. As a result the emission proceeds from electronic excited states which are not at equilibrium.
Similar content being viewed by others
References
Andreeva NA, Chermensky DN, Bezborodov AM (1978) Purification of extracellular trypsin inhibitor from Actinomyces janthinus 118. Appl Biochem Microbiol (Moscow) 14:25–31
Bakhshiev NG (1972) Spectroscopy of intermolecular interactions. Nauka, Leningrad
Bakhshiev NG, Mazurenko YuT, Piterskaya IV (1966) Luminescence decay in different portions of the luminescence spectrum of molecules in viscous solutions. Opt Spectrosc (USSR) 21:550–554
Bezborodova SI, Khodova OM, Stepanov VM (1983) Primary structure of Aspergillus clavatus ribonuclease C 2. Bioorg Chem (Moscow) 28:1136–1139
Burstein EA (1976) Luminescence of protein chromophores (Model studies). Ser Biophysica, vol 6. VINITI, Moscow
Burstein EA (1977) Intrinsic luminescence of proteins (origin and applications). Ser Biophysica, vol 7. VINITI, Moscow
Burstein EA (1983) Intrinsic protein luminescence as a method of studying rapid structural dynamics. Mol Biol (Moscow) 17:455–467
Burstein EA, Permyakov EA, Yashin VA, Burkhanov SA, Finazr-Argo A (1977) The fine structure of luminescence spectra of azurin. Biochim Biophys Acta 491:155–159
Demchenko AP (1981) Dependence of human serum albumin fluorescence spectrum on excitation wavelength. Ukr Biochim Z 53:22–27
Demchenko AP (1982) On the nanosecond mobility in proteins. Edge excitation fluorescence red shift of protein-bound 2(p-toluidinylnaphthalene)-6-sulfonate. Biophys Chem 15: 101–109
Demchenko AP (1984) Structural relaxation in protein molecules studied by fluorescence spectroscopy. J Mol Struct 114: 45–48
Demchenko AP (1985) Fluorescence molecular relaxation studies of protein dynamics. The fluorescence probe binding site of melittin is rigid on nanosecond timescale. FEBS Lett 182: 99–102
Demchenko AP (1986) Ultraviolet spectroscopy of proteins. Springer, Berlin Heidelberg New York
Demchenko AP, Ladokhin AS (1988) Red-edge-excitation fluorescence spectroscopy of indole and tryptophan. Eur Biophys J 15:369–379
Demchenko AP, Shcherbatska NV (1985) Nanosecond dynamics of the charged fluorescent probes at the polar interface of membrane phospholipid bilayer. Biophys Chem 22: 131–143
Eftink MR, Ghiron CA (1977) Exposure of tryptophanyl residues in proteins. Quantitative determination of fluorescence quenching studies. Biochemistry 16:5546–5551
Filenko AM, Zyma VL (1981) Two-wavelengths method for protein fluorescence small spectral shift registration. Mol Genet Biophys (Kiev) 6:126–135
Galley MC, Purkey RM (1970) Role of heterogeneity of the solvation site in electronic spectra in solution. Proc Natl Acad Sci USA 67:1116–1121
Grinvald A, Steinberg IL (1974) Fast relaxation processes in a protein revealed by the decay kinetics of tryptophan fluorescence. Biochemistry 13:5170–5177
Hazan G, Haas E, Steinberg IZ (1976) The fluorescence decay of human serum albumin and its subfractions. Biochim Biophys Acta 434:144–153
Hershberger MV, Maki AH, Galley WC (1980) Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins. Biochemistry 19:2204–2209
Inoue J, Sase S, Rhujo R, Nagaoka S, Sogami M (1979) Interaction between bovine plasma albumin and sodium dodecyl sulfate studied by means of 13C-NMR spectra. Biopolymers 18:373–382
Ivkova MN, Vedenkina NS, Burstein EA (1971) Fluorescence of tryptophan residues of serum albumin. Mol Biol (Moscow) 5:214–224
Kamalyan MG, Nalbandyan RM (1977) Optical and magnetic properties of azurin from Pseudomonas aeruginosa. Biochimia (Moscow) 42:223–229
Konev SV (1967) Fluorescence and phosphorescence of proteins and nucleic acids. Plenum Press, New York
Kuntz ID Jr, Kauzmann W (1974) Hydration of proteins and polypeptides. Adv Protein Chem 28:239–345
Lakowicz JR (1983) Principles of fluorescence spectroscopy. Plenum Press, New York London
Lakowicz JR, Balter A (1982) Direct recording of the initially excited and the solvent relaxed fluorescence emission spectra of tryptophan by phase sensitive detection of fluorescence. Photochem Photobiol 36:125–132
Lakowicz JR, Cherek H, Bevan DR (1980) Demonstration of nanosecond dipolar relaxation in biopolymers by inversion of apparent fluorescence phase shift and demodulation lifetimes. J Biol Chem 255:4403–4406
Longworth JW (1971) Luminescence of polypeptides and proteins. In: Steiner RF, Weinryb I (eds) Excited states of proteins and nucleic acids. Plenum Press, New York London, pp 319–487
Ludescher RD, Volwerk JJ, de Haas GH, Hudson BS (1985) Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A 2, its zymogen and an enzyme/ micelle complex. Biochemistry 24:7240–7250
Lumry R, Hershberger M (1978) Status of indole photochemistry with special reference to biological application. Photochem Photobiol 27:819–840
Macgregor RB, Weber G (1981) Fluorophores in polar media. Spectral effects of the Langevin distribution of electrostatic interactions. Ann NY Acad Sci 366:140–154
Maulet Y, Mathey-Prevot B, Kaiser Y, Ruegy UT, Fulpius BW (1980) Purification and chemical characterization of melittin and acetylated derivatives. Biochim Biophys Acta 625: 274–280
Mazurenko YuT, Bakhshiev NG (1970) The influence of orientational dipolar relaxation on spectral, temporal and polarizational properties of luminescence in solutions. Opt Spectrosc (USSR) 28:905–913
Nyamaa D, Bat-Erdene O, Burstein EA (1985) The medium effects on functional and structural properties of serum albumin. III. Effect of temperature and ionic strength on the N 1 − F, F 1 − F 2 and F 2 − E transitions of human serum albumin. Biophysica (Moscow) 19:833–840
Permyakov EA, Burstein EA (1975) Relaxation processes in frozen aqueous solution of proteins: temperature dependence of fluorescence parameters. Stud Biophys 51:91–103
Permyakov EA, Ostrovsky AV, Burstein EA, Pleashanov PG, Gerday Ch (1985) Parvalbumin conformers revealed by steady-state and time-resolved fluorescence spectroscopy. Arch Biochem Biophys 240:781–791
Rubinov AN, Tomin VI (1970) Bathochromic luminescence in low-temperature solutions of dyes. Opt Spectrosc (USSR) 29:1082–1086
Rubinov AN, Tomin VI (1984) Inhomogeneous broadening of electronic spectra of organic molecules in solid and liquid solutions. Preprint N 348, Institute of Physics, Minsk USSR
Teale FWJ (1960) The ultraviolet fluorescence of proteins in neutral solutions. Biochem J 76:381–388
Terwilliger TC, Weissman L, Eisenberg D (1982) The structure of mellitin in the form I crystals and its application for melittin's lytic and surface activities. Biophys J 37:353–361
Turoverov KK, Kuznetsovy IM, Zaitsev VN (1984) Azurin UV-Fluorescence interpretation on the basis of X-ray data. Bioorgan Chem (Moscow) 10:792–806
Vladimirov YuA, Burstein EA (1960) Luminescence spectra of aromatic amino acids and proteins. Biophysica (Moscow) 5:385–392
Weber G, Shinitzky M (1970) Failure of energy transfer between identical aromatic molecules on excitation of the longwave edge of the absorption spectrum. Proc Natl Acad Sci USA 65:823–830
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Demchenko, A.P. Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins. Eur Biophys J 16, 121–129 (1988). https://doi.org/10.1007/BF00255522
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00255522