Abstract
The dipolar relaxation process induced around tryptophan, indole and tyrosine in viscous media, as well as in several single tryptophan-containing proteins (staphylococcal nuclease, ribonuclease T1, melittin and albumin), has been studied by dynamic fluorescence measurements. A new theoretical model has been developed, including the relaxation dynamics directly in the fluorescence decay function. The phase shift and demodulation data have been fitted with this new algorithm which allows to resolve the different relaxation times influencing the fluorophore excited state. These parameters are in a good agreement with those measured with the traditional time-resolved emission spectroscopy. The results indicate that indeed a correlation exists between the radiative rate change obtained with the new model and the temporal spectral shift reported in the literature. Finally, this new approach has also been extended to the case of superoxide dismutase and phosphofructokinase, allowing to measure the relaxation time even in proteins lacking a temporal spectral shift during the fluorphore's lifetime.
Similar content being viewed by others
References
J. R. Lakowicz (2000). Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic, New York.
J. B. A. Ross, W. R. Laws, K. W. Rousslang, and H. R. Wyssbrod (1992). in J. R. Lakowicz (Ed.), Topics in Fluorescence Spectroscopy, Vol. 3, Plenum Press, New York, chap. 1.
C. Eggeling, S. Berger, L. Brand, J. R. Fries, J. Schaffer, A. Volkmer, and C. A. Seidel, (2001). Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. J. Biotechnol. 86, 163-180.
L. S. Slater and P. R. Callis (1995). Molecular orbital theory of the 1La ans 1Lb states of indole. 2. An ab initio study. J. Phys. Chem. 99, 8572-8581.
A. P. Demchenko (1986). Ultraviolet Spectroscopy of Proteins, Springer-Verlag, Berlin.
M. Vincent, J. Gallay, and A. P. Demchenko (1997). Dipolar relaxation around indole as evidenced by fluorescence lifetime distributions and time-dependence spectral shifts. J. Fluoresc. 7, 107S-110S.
A. S. Ladokhin (1999). Red-edge excitation study of non-exponential fluorescence decay of indole in solution and in a protein. J. Fluorescesc. 9, 1-10.
J. R. Lakowicz (2000). On spectral relaxation in proteins. Photochem. Photobiol. 72, 421-437.
B. De Foresta, J. Gallay, J. Sopkova, P. Champeil, and M. Vincent (1999). Tryptophan octyl ester in detergent micelles of dodecylmaltoside: Fluorescence properties and quenching by brominated detergent analogs. Biophys. J. 77, 3071-3084.
H. A. Garda, A. M. Bernasconi, R. R. Brenner, F. Aguilar, M. A. Soto, and C. P. Sotomayor (1997). Effect of polyunsaturated fatty acid deficiency on dipole relaxation in the membrane interface of rat liver microsomes. Biochim. Biophys. Acta 1323,97-104.
L. A. Bagatolli, T. Parasassi, G. D. Fidelio, and E. Gratton (1999). A model for the interaction of 6-lauroyl-2-(N,N dimethylamino)naphthalene with lipid environments: Implications for spectral properties. Photochem. Photobiol. 4, 557-564.
S. S. Antollini, M. A. Soto, I. Bonini de Romanelli, C. Gutierrez-Merino, P. Sotomayor, and F. J. Barrantes (1996). Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer. Biophys. J. 70, 1275-1284.
G. Mei, A. Di Venere, F. De Matteis, and N. Rosato (2003). The recovery of dipolar relaxation times from fluorescence decays as a tool to probe local dynamics in single tryptophan proteins. Archives Biochem. Biophys. 417, 159-164.
G. Mei, N. Rosato, N. Silva, R. Rusch, E. Gratton, I. Savini, and A. Finazzi Agrò (1992). Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: A dynamic fluorescence study. Biochemistry 31, 7224-7230.
E. Gratton, and M. Limkeman (1983). A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys. J. 44, 315-325.
G. Mei, A. Di Venere, F. De Matteis, A. Lenzi, and N. Rosato (2001). Asymmetrical distribution of intrinsic fluorescence lifetimes in proteins. J. Fluorescence 11, 319-333.
J. R. Alcala, E. Gratton, and F. G. Prendergast (1987). Fluorescence lifetime distributions in proteins. Biophys. J. 51, 597-604.
J.R. Lakowicz, H. Cherek, I. Gryczynski, N. Joshi, and M.L. Johnson (1987). Analysis of fluorescence decay kinetics measured in the frequency domain using distributions of decay times. Biophys. Chem. 28,35-50.
Ware, W. R. (1992). in V. Ramamurthy (Ed.), Photochemistry in organized and costrained media, VCA Publisher, New York, chap. 13.
A. G. Szabo, and D. M. Rayner (1980). Fluorescence decay of tryptophan conformers in aqueous solution. J. Am. Chem. Soc. 102, 554-563.
J. W. Petrich, M. C. Chang, D. B. McDonald, and G. R. Fleming (1983). On the origin of nonexponential fluorescence decay in tryptophan and its derivatives. J. Am. Chem. Soc. 105, 3824-3836.
R. F. Chen, J. R. Knutson, H. Ziffer, and D. Porter (1991). Fluorescence of tryptophan dipeptides: Correlations with the rotamer model. Biochemistry 30, 5184-5195.
B. Liu, R. K. Yhalji, P. D. Adams, F. R. Fronczek, M. L. McLaughlin, and M. D. Barkley (2002). Fluorescence of cis-1-Amin-2-(3-indolyl)cyclohexane-1-carboxylic acid: A single tryptophan χ1 rotamer model. J. Am. Chem. Soc. 124, 13329-13338.
M. R. Eftink, Y. Jia, D. Hu, and C. A. Ghiron (1995). Fluorescence studies with tryptophan analogs: excited state interactions involving the side chain amino group. J. Phys. Chem. 99, 5713-5723.
J. R. Lakowicz, and H. C. Cherek (1980). Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence. J. Biol. Chem. 255, 831-834.
A. P. Demchenko, and A. S. Ladokhin (1988). Red-edge-excitation fluorescence spectroscopy of indole and tryptophan. Eur. Biophys. J. 15, 369-379.
J.R. Lakowicz, H. Szmacinski, and I. Gryczynski (1988). Picosecond resolution of indole anisotropy decays and spectral relaxation by 2 GHz frequency-domain fluorometry. Photochem. Photobiol. 47, 31-41.
D. Toptygin and L. Brand (2000). Spectrally-and time-resolved fluorescence emission of indole during solvent relaxation: A quantitative model. Chem. Phys. Lett. 322, 496-502.
E. J. P. Malar and K. Jug (1984). Structures and properties of excited states of benzene and some monosubstituted benzenes. J. Phys. Chem. 88, 3508-3516.
A. P. Demchenko (1988). Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins. Eur. Biophys. J. 16, 121-129.
P. R. Callis (1997). 1La and 1Lb transitions of tryptophan: Applications of theory and experimental observations to fluorescence of proteins. Methods Enzymol. 278, 113-150.
M.R. Eftink, I. Gryczynski, W. Wiczk, G. Laczko, and J.R. Lakowicz (1991). Effects of temperature on the fluorescence intensity and anisotropy decays of staphylococcal nuclease and the less stable nuclease-conA-SG28 mutant. Biochemistry 30, 8945-8653.
A. J. W. G. Visser, J. van Engelen, N. V. Visser, A. van Hoek, R. Hilhorst, and R. B. Freedman (1994). Fluorescence dynamics of staphylococcal nuclease in aqueous solution and reversed micelles. Biochim. Biophys. Acta 1204, 225-234.
A. P. Demchenko, I. Gryczynsky, Z. Gryczynsky, W. Wiczk, H. Malak, and M. Fishman (1993). Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease. Biophys. Chem. 48,39-48.
M. Eftinkand C.A. Ghiron (1987). Frequencydomainmeasurements of the fluorescence lifetime of ribonuclease T1. Biophys. J. 52, 467-473.
I. Gryczynski, M. Eftink, and J. R. Lakowicz (1988). Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1. Biochim. Biophys. Acta 954, 244-252.
J. R. Lakowicz, B. P. Maliwal, H. Cherek, and A. Balter (1983). Rotational freedom of tryptophan residues in proteins and peptides. Biochemistry 22, 1741-1752.
U. Heinemann and W. Saenger (1982). Specific protein-nucleic acid recognition in ribonuclease T1-2-guanylic acid complex: An X-ray study. Nature 299,27-31.
A. P. Demchenko and A. S. Ladokhin (1988). Temperature-dependent shift of fluorescence spectra without conformational changes in protein; studies of dipole relaxation in the melittin molecule. Biochim. Biophys. Acta 955, 352-360.
M. Fisz (1963). Probability Theory and Mathematical Statistics, Wiley, New York, chap. 12.
A. Buzády, J. Erostyák, and B. Somogyi (2000). Phase-fluorimetry study on dielectric relaxation of human serum albumin. Biophys. Chem. 88, 153-163.
S. J. Kim, F. N. Chowdhury, W. Stryjewski, E. S. Younathan, P. S. Russo, and M. D. Barkley (1993). Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase. Biophys. J. 65, 215-226.
N. Silva, E. Gratton, G. Mei, N. Rosato, R. Rusch, and A. Finazzi Agro' (1993). Molten globule monomers in human superoxide dismutase. Biophys. Chem. 48, 171-182.
Y. Chen and M. D. Barkley (1998). Toward understanding tryptophan fluorescence in proteins. Biochemistry 37, 9976-9982.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mei, G., Di Venere, A., Agrò, A.F. et al. Dipolar Relaxation Times of Tryptophan and Tyrosine in Glycerol and in Proteins: A Direct Evaluation from Their Fluorescence Decays. Journal of Fluorescence 13, 467–477 (2003). https://doi.org/10.1023/B:JOFL.0000008057.70137.c7
Issue Date:
DOI: https://doi.org/10.1023/B:JOFL.0000008057.70137.c7