Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state atpH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80×102 M−1 sec−1 for trypsin-inhibiting domain and 0.77× 102 M−1 sec−1 for subtilisin-inhibiting domain of the inhibitor at 30°C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1–2%α-helix, 40–42%β-sheet, and 57–58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 Å (fully reduced inhibitor) to 18.8 Å (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway.
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Baldwin, R. L. (1993).Curr. Opin. Struct. Biol. 3, 84–91.
Corbett, R. J. T., and Roche, R. S. (1984).Biochemistry 23, 1888–1894.
Creighton, T. E. (1974).J. Mol. Biol. 87, 563–577.
Creighton, T. E. (1978).Prog. Biophvs. Mol. Biol. 33, 231–297.
Creighton, T. E. (1979).J. Mol. Biol. 129, 411–431.
Creighton, T. E. (1984).Meth. Enzymol. 107, 305–329.
Ellman, G. L. (1959).Arch. Biochem. Biophys. 82, 70–77.
Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961).Arch. Biochem. Biophys. 95, 271–278.
Grutler, M. G., Fendrich, G., Huber, R., and Bode, W. (1988).EMBO J. 7, 345–351.
Harrison, S. C., and Durbin, R. (1985).Proc. Natl. Acad. Sci. USA 82, 4028–4030.
Heinzel, R., Appelhans, H., Gassen, H. G., Seemuller, V., Arnhold, M., Fritz, H., Lottspeich, F., Widenmann, K., and Machleidt, W. (1987). InPulmonary Emphysema and Proteolysis: 1986 (Taylor, J. C., and Mittman, C., eds.), Academic Press, New York, pp. 297–306.
Hummel, B. C. W. (1959).Can. J. Biochem. Physiol. 37, 1393–1399.
Kassell, B., and Williams, M. J. (1976). InHandbook of Biochemistry and Molecular Biology (Fasman, G. D., ed.) CRC Press, Cleveland, Ohio, Vol. II, pp. 583–604.
Kato, I., and Tominaga, N. (1979).Fed. Proc. 33, 832.
Kim, P. S., and Baldwin, R. L. (1982).Annu. Rev. Biochem. 51, 459–489.
Kim, P. S., and Baldwin, R. L. (1990).Annu. Rev. Biochem. 59, 630–660.
Konisberg, W. (1972).Meth. Enzymol. 25B, 185–188.
Laskowski, M., Jr., Kato, I., Leary, T. R., Schrode, J., and Sealock, R. W. (1974). InBayer-Symposium V: Proteinase Inhibitors (Fritz, H., Tschesche, H., Greene, L. J., and Truscheit, E., eds.), Springer-Verlag, Berlin, pp. 597–611.
Levinthal, C. (1968).J. Chem. Phys. 65, 44–45.
Light, A., and Higaki, J. N. (1987).Biochemistry 26, 5556–5564.
Light, A., Higaki, J. N., and Odoxzynski, T. W. (1987). InProtein Structure, Folding and Design 2 (Oxender, D. L., ed.), Alan R. Liss, New York, pp. 341–351.
Ottenson, M., and Svendsen, I. (1970).Meth. Enzymol. 19, 199–205.
Ptitsyn, O. B. (1987).J. Protein Chem. 6, 273–293.
Reisfeld, R. E., Lewis, U. J., and Williams, D. E. (1962).Nature 195, 281–283.
Schmidt, F. X. (1992). InProtein Folding (Creighton, T. E., ed.), Freeman, New York, pp. 197–241.
Sil, P. C., Chaudhuri, T. K., and Sinha, N. K. (1993).J. Protein Chem. 12, 71–78.
Swank, R. T., and Munkres, K. D. (1971).Ann. Biochem. 39, 462–477.
Tamburro, A. M., Boccu, M., and Celotti, L. (1970).Int. J. Protein Res. 2, 157–164.
Thomson, R. E., Spivey, H. O., and Katz, A. J. (1976).Biochemistry 15, 862–867.
Woodward, C. K., and Rosenberg, A. (1970).Proc. Natl. Acad. Sci. USA 66, 1067–1074.
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Chaudhuri, T.K., Sinha, N.K. Refolding of trypsin-subtilisin inhibitor from marine turtle eggwhite. J Protein Chem 15, 315–320 (1996). https://doi.org/10.1007/BF01887120
- Protein folding
- protein structure
- trypsin-subtilisin inhibitor
- marine turtle eggwhite