Conformational Changes at the Active Site of D-Glyceraldehyde-3-Phosphate Dehydrogenase During Unfolding in Guanidine Solution
The fluorescent NAD derivative, covalently linked to the active site sulfhydryl groups of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (Tsou, C.L. et al., Biochem. Soc. Trans. 11, 425–429, 1983), has been used as a probe to study the conformational change at the active site of this enzyme during unfolding by guanidine denaturation. This derivative shows both a red shift of its emission maximum and a decrease in fluorescence intensity at the same guanidine concentration which brought about complete inactivation together with similar changes of the intrinsic protein fluorescence. Complete unfolding of the enzyme as indicated by further red shift in the emission maximum and decrease in intensity of the intrinsic fluorescence requires much higher guanidine concentrations. It appears that the low guanidine concentrations required to bring about complete inactivation also lead to perturbation of the active site conformation and that a Trp residue is situated at or near the active site region.
KeywordsCreatine Kinase Emission Intensity Emission Maximum Intrinsic Fluorescence Guanidine Hydrochloride
Unable to display preview. Download preview PDF.
- 1.Thomas, J.O. (1974) in: Companion to Biochemistry (Bull, A.T., Lagnado, J.R., Thomas, J.O. and Tipton, K.F., eds.) pp. 87–138, Longman Green, LondonGoogle Scholar
- 4.Lundbladd, R.L. (1984) Chemical Reagents for Protein Modification Vol. 1, pp. 1–23, CRC Press, ClevelandGoogle Scholar
- 10.Yao, Q.Z., Zhou, H.M., Hou, L.X. and Tsou, C.L. (1982) Sci. Sin. 25B, 1296–1302Google Scholar
- 11.Yao, Q.Z., Tian, M. and Tsou, C.L. (1985) Sci. Sin. 28B, 484–493Google Scholar
- 12.Tsou, C.L., Xu, G.Q., Zhou, J.M. and Zhao, K.Y. (1983) Biochem. Soc. Trans. 11, 425–429Google Scholar
- 13.Ho, Y. S., Lian, Y.N., Chiang, S.H. and tTsou, C.L. (1979) Sci. Sin. 22, 207–221Google Scholar
- 14.Tsou, C.L., Zhao, K.Y. and Ho, Y.S. (1979) Sci. Sin. 22, 693–704Google Scholar
- 15.Ho, Y.S. and Tsou, C.L. (1979) Sci. Sin. 22, 478–489Google Scholar
- 16.Lian, Y.N. and Tsou, C.L. (1981) Sci. Sin. 24, 1146–1153Google Scholar
- 18.Heilmann, H.D. and Pfleiderer, G. (1975) Biochim. Biophys. Acta 384, 331–341Google Scholar
- 19.Moras, D., Olsen, K.W., Sabesen, M.N., Buehner, M., Ford, G.C. and Rossmann, M.G. (1975) J. Biol. Chem. 250, 9137–9162Google Scholar
- 21.Ho, Y.S., Liang, S.J. and Tsou, C.L. (1980) Biochim. Biophys. Acta 613, 249–255Google Scholar