The refolding of denatured rabbit muscle creatine kinase.
1. The refolding of rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) which had been denatured in 3 M guanidine hydrochloride was monitored by studying the regain of enzyme activity. Full activity could be regained provided that the residual denaturant concentration was less than or equal to 0.1 M. 2. The refolded product was shown by a number of criteria (CD, kinetic parameters and polyacrylamide gel electrophoresis) to be identical with the native enzyme. 3. The rate of regain of enzyme activity was studied as a function of protein concentration. It was found that 70% of the activity was regained in a rapid, first-order process. The remaining activity was regained more slowly. In the rapid phase the number of reactive thiol groups per subunit declined from four to two; the further decline to one per subunit occurred more slowly. 4. It was found that the presence of the reducing agent dithiothreitol was not necessary for the regain of full activity, provided that the chelating agent EDTA was present. 5. The subunit structure of the enzyme during refolding was studied using dimethylsuberimidate as a cross-linking agent. From these experiments, a pathway for the refolding process could be proposed.
D000818:Animals; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000032:analysis*; D004120:Dimethyl Suberimidate; D004229:Dithiothreitol; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006146:Guanidines; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011489:Protein Denaturation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
D006146:Guanidines; D013438:Sulfhydryl Compounds; D004120:Dimethyl Suberimidate; D004492:Edetic Acid; D003402:Creatine Kinase; D004229:Dithiothreitol