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Protein Fluorescence

  • Chapter
Principles of Fluorescence Spectroscopy

Abstract

Extensive data have been published on the fluorescence of proteins and peptides. This emphasis is a result of the presence of natural fluorophores in almost all proteins, these being tyrosine and tryptophan. The fluorescence of most proteins is dominated by the tryptophan residues, and the indole nucleus of these residues is a uniquely sensitive and complicated fluorophore. Indole, tryptophan, and their derivatives are highly sensitive to solvent polarity, and appear to be subject to both general and specific solvent effects. As a result, the emission spectra of tryptophan residues can reflect the polarity of their surrounding environment. The emission spectra of proteins are sensitive to the binding of substrates, association reactions, and to denaturation. Polarization measurements of tryptophan residues reflect the average correlation time of these residues, despite the experimental difficulties posed by their complex polarization spectrum. Hence, polarization measurements can reveal the effects of denaturants on protein structure and the association reactions of proteins with ligands and with other macromolecules. Finally, tryptophan appears to be uniquely sensitive to quenching by a variety of substances. In addition to quenching by oxygen and iodide, tryptophan is quenched by substances such as acrylamide, succinimide, hydrogen peroxide, dichloroacetamide, pyridinium hydrochloride, cysteine, chlorinated hydrocarbons, \( NO_3^ - \), \( I0_3^ - \), Cs2+, Cu2+, Pb2+, Cd2+, and Mn2+.(1,2) This sensitivity to a variety of quenchers appears to be a result of a propensity of the excited indole nucleus to donate electrons while in the excited state. The sensitivity to quenchers allows determination of the accessibility of the tryptophan residues in proteins by quenching measurements.

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References

  1. Eftink, M. R., and Ghiron, C. A., 1981, Fluorescence quenching studies with proteins, Anal. Biochem. 114:199–227.

    Article  CAS  Google Scholar 

  2. Steiner, R. F., and Kirby, E. P., 1969, The interaction of the ground and excited state of indole derivatives with electron scavengers, J. Phys. Chem. 73:4130–4135.

    Article  CAS  Google Scholar 

  3. Konev, S. V., 1967, Fluorescence and Phosphorescence of Proteins and Nucleic Acids, Plenum Press, New York.

    Book  Google Scholar 

  4. Weinryb, I., and Steiner, R. F. (Eds.), 1971, Excited States of Protein and Nucleic Acids, Plenum Press, New York.

    Google Scholar 

  5. Chen, R. F., and Edelhoch H. (Eds.), 1976, Biochemical Fluorescence, Vol. 2 Concepts, Marcel Dekker, New York.

    Google Scholar 

  6. Wetlaufer, D. B., 1962, Ultraviolet absorption spectra of proteins and amino acids, Advan. Protein Chem. 17:303–390.

    Article  CAS  Google Scholar 

  7. Teale, F. W. J., and Weber, G., 1957, Ultraviolet fluorescence of the aromatic amino acids,Biochem. J. 65:476–482.

    CAS  Google Scholar 

  8. Weber, G., 1960, Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan, and related compounds, Biochem. J. 75:335–345.

    CAS  Google Scholar 

  9. Valeur, B., and Weber, G., 1977, Resolution of the fluorescence excitation spectrum of indole into the 1 L a and 2 L b excitation bands, Photochem. Photobiol. 25:441–444.

    Article  CAS  Google Scholar 

  10. Strickland, E. H., Horwitz, J., Kay, E., Shannon, L. M., Wilchek, M., and Billups, L., 1971, Near-ultraviolet absorption bands of tryptophan. Studies using horseradish peroxidase isoenzymes, bovine and horse heart cytochrome c, and AT-stearoyl-L-tryptophan /t-hexyl ester, Biochemistry 10:2631–2638.

    Article  CAS  Google Scholar 

  11. Walker, M. S., Bednar, T. W., and Lumry, R., 1966, Exciplex formation in the excited state, J. Chem. Phys. 45:3455–3456.

    Article  CAS  Google Scholar 

  12. Hershberger, M. V., Lumry, R., and Verral, R., 1981, The 3-methylindole/n-butanol exciplex: Evidence for two exciplex sites in indole compounds, Photochem. Photobiol. 33:609–617.

    Article  CAS  Google Scholar 

  13. Vladimirov, Y. A., and Burstein, E. A., 1960, Luminescent spectra of aromatic amino acids and proteins, Biophysika 5:385–392.

    CAS  Google Scholar 

  14. Eisinger, J., 1969, Intramolecular energy transfer in adrenocorticotropin,Biochemistry 8:3902–3908.

    Article  CAS  Google Scholar 

  15. Teale, F. W. J., 1960, The ultraviolet fluorescence of proteins in neutral solution,Biochem. J. 76:381–388.

    CAS  Google Scholar 

  16. Edelhoch, H., Perlman, R. L., and Wilchek, M., 1968, Fluorescence studies with tyrosyl peptides, Biochemistry 7:3893–3900.

    Article  CAS  Google Scholar 

  17. Weber, G., and Rosenheck, K., 1964, Proton-transfer effects in the quenching of fluorescence of tyrosine copolymers, Biopolymers Symp. 1:333–344.

    Google Scholar 

  18. Cowgill, R. W., 1976, “Tyrosyl fluorescence in proteins and model peptides,” in Biochemical Fluorescence Concepts, R. F. Chen and H. Edelhoch (Eds.), Marcel Dekker, New York, pp. 441–486.

    Google Scholar 

  19. Saito, Y., Tachibana, H., Hayaski, H., and Wada, A., 1981, Excitation energy transfer between tyrosine and tryptophan in proteins evaluted by the simultaneous measurement of fluorescence and absorbance, Photochem. Photobiol. 33:289–295.

    Article  CAS  Google Scholar 

  20. Longworth, J. W., and Ghiron, C. A., 1979, Transfer of singlet energy within trypsin, Biochemistry 18:3828–3832.

    Article  Google Scholar 

  21. Kronman, M. J., and Holmes, L. G., 1971, The fluorescence of native, denatured, and reduced denatured protein, Photochem. Photobiol. 14:113–134.

    Article  CAS  Google Scholar 

  22. Eftink, M. R., and Ghiron, C. A., 1976, Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies,Biochemistry 15:672–680.

    Article  CAS  Google Scholar 

  23. Burstein, E. A., Permyakov, E. A., Yashion, V. A., Burkhanov, S. A., and Finazz Agro, A., 1977, The fine structure of luminescence spectra of azurin, Biochem. Biophys. Acta 491:155–159.

    CAS  Google Scholar 

  24. Brand, J. G., and Cagen, R. H., 1977, Fluorescence characteristics of native and denatured monellin, Biochim. Biophys. Acta. 493:178–187.

    CAS  Google Scholar 

  25. Cagen, R. H., Brand, J. G., Morris, J. A., and Morris, R. W., 1978, Monellin, a sweet-tasting protein, Sweetness Dental Caries, 311–323.

    Google Scholar 

  26. Talbot, J. C., Dufourcq, J., de Bong, J., Faucon, J. R., and Lurson C., 1979, Conformational change and self association of monomeric melittin, FEBS Lett. 102:191–193.

    Article  CAS  Google Scholar 

  27. Faucon, J. F., Dufourcq, J., and Lussan, C., 1979, The self association of melittin and its binding to lipids, FEBS Lett. 102:187–190.

    Article  CAS  Google Scholar 

  28. Georghiou, S., Thompson, M., and Mukhopadhyay, A. K., 1981, Melittin-phospholipid interactions. Evidence for melittin aggregation, Biochim. Biophys. Acta 642:429–432.

    Article  CAS  Google Scholar 

  29. Shimizu, O., and Imakuvo, K., 1977, New emission band of tyrosine induced by interaction with phosphate ion, Photochem. Photobiol. 26:541–543.

    Article  CAS  Google Scholar 

  30. Behmaarai, T. A., Toulme, J. J., and Helene, C., 1979, Quenching of tyrosine fluorescence by phosphate ions. A model study for protein-nucleic acid complexes, Photochem. Photobiol. 30:533–539.

    Article  Google Scholar 

  31. Szabo, A., Lynn, K. R., Krajcarski, D. T., and Rayner, D. M., 1978, Tyrosinate fluorescence maxima at 345 nm in proteins lacking tryptophan at pH 7,FEBS Lett. 94:249–252.

    Article  CAS  Google Scholar 

  32. Grinvald, A., and Steinberg, I. Z., 1976, The fluorescence decay of tryptophan residues in native and denatured proteins,Biochem. Biophys. Acta 427:663–678.

    CAS  Google Scholar 

  33. Rayner, D. M., and Szabo, A. G., 1978, Time-resolved fluorescence of aqueous tryptophan, Can. J. Chem. 56:743–745.

    Article  CAS  Google Scholar 

  34. Szabo, A. G., and Rayner, D. M., 1980, Fluorescence decay of tryptophan conformers in aqueous solution, J. Amer. Chem. Soc. 102:554–563.

    Article  CAS  Google Scholar 

  35. Ross, J. A. B., Rousslang, K. W., and Brand, L., 1981, Time-resolved fluorescence and anisotropy decay of the tryptophan in adrenocorticotropin (1–24), Biochemistry 20:4361–4369.

    Article  CAS  Google Scholar 

  36. Cockle, S. A., and Szabo, A. G., 1981, Time-resolved fluorescence spectra of tryptophan in monomeric glucagon, Photochem. Photobiol 34:23–27.

    CAS  Google Scholar 

  37. Lakowicz, J. R., and Cherek, H., 1980, Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence, J. Biol. Chem. 255:831–834.

    CAS  Google Scholar 

  38. Lakowicz, J. R., and Baiter, A., 1982, Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays, Biophys. Chem. 15:353–360.

    Article  CAS  Google Scholar 

  39. Ross, J. A., Schmidt, C. J., and Brand, L., 1981, Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase, Biochemistry 20:4369–4377.

    Article  CAS  Google Scholar 

  40. Brochon, J. C., Wahl, Ph., Charlier, M., Maurizat, J. C., and Helene, C., 1977, Timeresolved spectroscopy of the tryptophanyl fluorescence of the E. coli Lac repressor, Biochem. Biophys. Res. Commun. 79:1261–1271.

    Article  CAS  Google Scholar 

  41. Privat, J. P., Wahl, P., Auchet, J. C., and Pain, R. H., 1980, Time-resolved spectroscopy of tryptophyl fluorescence of yeast 3-phosphoglycerate kinase, Biophys. Chem. 11:239–248.

    Article  CAS  Google Scholar 

  42. Lakowicz, J. R., and Maliwal, B., 1982, Unpublished observation.

    Google Scholar 

  43. DeLauder, W. B., and Wahl, Ph., 1971, Effect of solvent upon the fluorescence decay of indole,Biochem. Biophys. Acta. 243:153–163.

    CAS  Google Scholar 

  44. Privat, J. P., Wahl, P., and Auchet, J. C., 1979, Rates of deactivation processes of indole in water-organic solvent mixtures—Application to tryptophanyl fluorescence of proteins, Biophys. Chem. 9:223–233.

    Article  CAS  Google Scholar 

  45. Burstein, E. A., Vedenkina, N. S., and Ivkova, M. N., 1973, Fluorescence and the location of tryptophan residues in protein molecules, Photochem. Photobiol. 18:263–279.

    Article  CAS  Google Scholar 

  46. Richards, F. M., 1974, The interpretation of protein structure: Total volume, group volume distributions, and packing density, J. Mol. Biol. 82: 1–14.

    Article  CAS  Google Scholar 

  47. Lakowicz, J. R., and Weber, G., 1973, Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond timescale, Biochemistry 12:4171–4179.

    Article  CAS  Google Scholar 

  48. Weber, G., and Shinitzky, M., 1970, Failure of energy transfer between identical aromatic molecules on excitation at the long wavelength edge of the absorption spectrum, Proc. Natl. Acad. Sci. 65:823–830.

    Article  CAS  Google Scholar 

  49. Eftink, M. R., and Ghiron, C. A., 1977, Exposure of tryptophanyl residues and protein dynamics, Biochemistry 16:5546–5551.

    Article  CAS  Google Scholar 

  50. Munro, I., Pecht, I., and Stryer, L., 1979, Subnanosecond motions of tryptophan residues in proteins, Proc. Natl. Acad. Sci. 76:56–60.

    Article  CAS  Google Scholar 

  51. Lakowicz, J. R., Maliwal, B., Cherek, H., and Baiter, A., 1983, Dynamics of tryptophan residues in proteins quantified by lifetime-resolved anisotropics, Biochemistry (in press).

    Google Scholar 

  52. Mantulin, W. W., and Weber, G., 1977, Rotational anisotropy and solvent-fluorophore bonds: An investigation by differential polarized phase fluorometry, J. Chem. Phys. 66:4092–4099.

    Article  CAS  Google Scholar 

  53. Grinvald, A., and Steinberg, I. Z., 1974, Fast relaxation processes in a protein revealed by the decay kinetics of tryptophan fluorescence, Biochemistry 13:5170–5178.

    Article  CAS  Google Scholar 

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  1. University of Maryland School of Medicine, Baltimore, Maryland, USA

    Joseph R. Lakowicz

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  1. Joseph R. Lakowicz
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© 1983 Plenum Press, New York

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Lakowicz, J.R. (1983). Protein Fluorescence. In: Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-7658-7_11

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  • DOI: https://doi.org/10.1007/978-1-4615-7658-7_11

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4615-7660-0

  • Online ISBN: 978-1-4615-7658-7

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