Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine.
Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.
Laboratory of Molecular Enzymology, Institute of Biophysics, Academia Sinica, Beijing, China.
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D000465:Algorithms; D000818:Animals; D002417:Cattle; D003568:Cytidine Monophosphate / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012996:Solutions; D013329:Structure-Activity Relationship
D006146:Guanidines; D012996:Solutions; D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate; D019791:Guanidine