The influence of effectors on the refolding (reactivation) of immobilized trypsin.
The refolding of immobilized trypsin in the presence of various effectors of its enzymatic activity has been studied. Trypsin covalently bound to Sephadex G-200 was made to unfold in a concentrated solution of urea; at the same time its S-S bonds were split with the help of dithiothreitol. The preparation was then separated from the splitting agents, and one of the effectors of the enzymatic activity of trypsin (boric acid, benzamidine, pancreatic or soybean inhibitors of trypsin, N-benzoyl-L-arginine ethyl ester, and N-tosyl-L-arginine methyl ester) was added and the reactivation of immobilized enzyme was studied in the absence of catalysts of thiol-disulphide exchange. The following effects were found. 1. The reactivation of immobilized trypsin in the presence of specific substrates or protein inhibitors proceeds with the same yield (2-5%) as in their absence. 2. In the presence of benzamidine or boric acid (competitive inhibitors) the reactivation yields of the immobilized trypsin increased 5-fold and 12-fold respectively, and became equal to 15% and 40%. Comparison of these results with the statistical probability of formation of the six native S-S bonds from twelve SH groups (approximately 0.01%) shows that if trypsin is made to refold in an immobilized state in the presence of a 'good' effector, the yield of the reactivation of the enzyme can be increased several thousand times. 3. Similar effects were observed for trypsin immobilized on Sepharose 4B. A model is suggested in terms of which the influence of various effectors on trypsin refolding is explained as being a result of their ability to bind with an intermediate folded forms of protein followed by a shift of the equilibrium towards 'regular' conformers.
D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides; D004793:Enzyme Reactivators / Q000494:pharmacology*; D004800:Enzymes, Immobilized; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D013438:Sulfhydryl Compounds; D014357:Trypsin / Q000032:analysis*
D004220:Disulfides; D004793:Enzyme Reactivators; D004800:Enzymes, Immobilized; D013438:Sulfhydryl Compounds; D014357:Trypsin