Biochemistry zyxwvu 1984, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC 23, zyxwvu 3891-3899 3891 Finney, J. L. (1979) in Water: A Comprehensive Treatise (Franks, F., Ed.) Vol. 6, pp 47-120, Plenum Press, New York, London. Gelin, B. R., zyxwvutsrqp & Karplus, M. (1979) Biochemistry zyxwvu 18, Haddad, L. C., Thayer, W. S., & Jenkins, W. T. (1977) Arch. Hess, G. P. (1971) Enzymes, 3rd Ed. 3, 213-248. Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C., & Ropley, J. A. (1972) Enzymes, 3rd Ed. 7, 665-868. Jenkins, W. T., & D'Ari, L. (1966) J. Biol. Chem. 241, 5667-5674. Johnson, F. A,, Lewis, zyxwvutsrqp S. D., & Shafer, J. A. (1981a) Bio- chemistry 20, 44-48. Johnson, F. A., Lewis, S. D., & Shafer, J. A. (1981b) Bio- chemistry 20, 52-58. Kirkwood, J. G. (1934) J. Chem. Phys. 2, 351-361. Kirkwood, J. G., & Westheimer, F. H. (1938) J. Chem. Phys. Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jon- sonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525. Laidler, K. J. (1978) Physical Chemistry with Biological Applications, pp 268-272, Benjamin/Cummings Co., Inc., Menlo Park, CA. Lewis, S. D., Johnson, F. A,, & Shafer, J. A. (1976) Bio- chemistry 15, 5009-5017. Lewis, S. D., Johnson, F. A,, & Shafer, J. A. (1981) Bio- chemistry 20, 48-5 1. 1256- 1268. Biochem. Biophys. 181, 66-72. 6, 506-512. Mehler, E. L. (1980) J. Am. Chem. SOC. 102, 4051-4056. Ohe, M., & Kajita, A. (1980) Biochemistry 19, 4443-4450. Owen, G. E. (1963) Electromagnetic Theory, Allyn and Ba- Parsons, S. M., & Raftery, M. A. (1972) Biochemistry 11, Pethig, R. (1979) Dielectric and Electronic Properties zy of Poe, M., Hoogsteem, K., & Matthews, D. A. (1979) J. Biol. Pollock, E. L., Alder, B. J., & Pratt, L. R. (1980) Proc. Natl. Rees, D. C. (1980) J. Mol. Biol. 141, 323-326. Schwarzenbach, G. (1936) 2. Phys. Chem. A 176, 133-153. Shire, S. J., Hanania, G. I. H., & Gurd, F. R. N. (1974a) Shire, S. J., Hanania, G. I. H., & Gurd, F. R. N. (1974b) Shire, S. J., Hanania, G. I. H., & Gurd, F. R. N. (1975) van Duijnen, P. Th., Thole, B. Th., & Hol, W. G. J. (1979) Warshel, A. (1979) Photochem. Photobiol. 30, 285-290. Warshel, A., & Levitt, M. (1976) J. Mol. Biol. 103, 227-249. Watenpaugh, K. D., Margulis, T. N., Sieker, L. C., & Jensen, Webb, T. J. (1926) J. Am. Chem. SOC. 48, 2589-2603. con, Boston. 1623-1629. Biological Materials, Wiley, Chichester. Chem. 254, 8143-8152. Acad. Sci. U.S.A. 77, 49-51. Biochemistry 13, 2967-2974. Biochemistry 13, 2974-2979. Biochemistry 14, 1352-1358. Biophys. Chem. 9, 273-280. L. H. (1978) J. Mol. Biol. 122, 175-190. Indole Fluorescence Quenching Studies on Proteins and Model Systems: Use of the Inefficient Quencher Succinimidet Maurice R. Eftink* and Camillo A. Ghiron ABSTRACT: We have compared the quenching of the fluorescence of proteins by acrylamide and succinimide, two chemically similar quenchers. We find that the ratio of the apparent rate constants for succinimide and acrylamide quenching, Y~/~, ranges from -0.1 to -0.7. Proteins having relatively buried tryptophan residues, such as ribonuclease T1, zyxwvu cod parvalbumin, and aldolase, are found to have small values of ySJA (i.e., succinimide quenches with a much smaller rate constant than acrylamide); proteins with relatively solvent- exposed tryptophan residues, such as glucagon and adreno- corticotropin, are found to have larger values of 7?lA. We interpret this range of Y ~ / ~ values as being due to either (a) a critical size dependence of the dynamic penetration of quencher through a protein matrix (succinimide being larger than acrylamide) and/or (b) an inherent dependence of the S t u d i e s of indole fluorescence quenching by added solutes have, in recent years, provided valuable information regarding the structure and dynamics of proteins in solution (Lehrer, From the Department of Chemistry, The University of Mississippi, University, Mississippi 38677 (M.R.E.), and the Department of Bio- chemistry, University of Missouri, Columbia, Missouri 65201 (C.A.G.). Received December 6, 1983. This work was supported by National Science Foundation Grants PCM-8206073 and PCM-7726614. A pre- liminary report of this work has appeared (Eftink & Ghiron, 1983). 0006-2960/84/0423-3891$01.50/0 succinimide quenching reaction on the microenvironment of the indole ring. The latter interpretation is supported by studies of the solvent dependence of the quenching of the fluorescence of indole and 5-methoxyindole by succinimide and acrylamide. These studies show that whereas acrylamide is an efficient quencher in all solvents investigated, succinimide is a relatively inefficient quencher in aprotic solvents. Thus, both of the above molecular bases for poor quenching of in- terior tryptophan residues in proteins by succinimide (Le., a critical size dependence of a microenvironment dependence) are consistent with the fluorescence quenching process oc- curring within the globular structure of proteins by a dynamic penetration mechanism, as opposed to an unfolding mechanism by which interior residues would be periodically exposed to the solvent. 1971; Lehrer & Leavis, 1978; Lakowicz & Weber, 1973; Lakowicz et al., 1983; Eftink & Ghiron, 1976a, 1977, 1981). The most significant finding with this technique is that even tryptophan (Trp) residues that are presumably deeply buried within globular proteins are able to be quenched by the un- charged quenchers oxygen and acrylamide with quenching rate constants on the order of lo9 M-' s-l. These results have been interpreted as indicating that the quenchers are able to pen- etrate into the matrix of globular proteins, with this penetration zyxwvutsrqp 0 1984 American Chemical Society