Formation of Enzymatically Active Intermediates during the Renaturation Process of Reduced Taka-amylase A*

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Abstract
Reduction of all 4 disulf ide bonds in Taka-amylase A by p-mercaptoethanol in 8 [image] urea yields a randomly coiled polypep-tide chain. Enzymatic activity can be recovered by removal of the reducing and denaturing reagents and by subsequent air-oxidation. The present investigation was attempted to make clear what kinds of molecular species are involved as the intermediate during the reoxi-dation process, and whether the reformation of all the intact disulfide bonds of Taka-amylase A is necessary for the recovery of the enzymatic activity. Reduced Taka-amylase A was partially oxidized to various levels and carboxymethylated. Intermediates differing in their sulfhy dryl content acquired different net charges by carboxymethylation and could be successfully separated into 4 components by gel-filtration and subsequent diethylaminoethyl-cellulose column chromatography. Among them, 3 components had enzymatic activity. One was fully renatured enzyme. The other 2 were the derivatives in which some sulfhydryl groups were partially reoxidized and the remaining residual sulfhydryl groups were carboxymethylated. These 2 components had regained 1 and 3 disulfide bonds per molecule, respectively. They had 5 and 55% of the original enzymatic activity, respectively. Partially reoxidized Taka-amylase A before carboxymethylation might have regained a higher enzymatic activity than the carboxymethylated Taka-amylase A. It was thus demonstrated that reformation of all the original disulfide bonds is not necessary for the recovery of enzymatic activity of Taka-amylase A. A scheme of the reoxidation process was presented.

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