The Activation of Papain

In previous reports (1, 2) there was presented evidence which could not be fitted into the widely accepted oxidation-reduction theory of papain activation (3). In these experiments it was shown that papain may be inhibited by means of phenylhydrazine and may be reactivated by means of benzaldehyde. In an attempt to study further the process of papain activation, an inactive papain preparation (Papain A) was activated by HCN (HCNPapain A) and subsequently precipitated by means of isopropyl alcohol. Th& activity of the enzyme preparation was determined before and after this precipitation. If the activation of papain by HCN consisted simply in the reduction of disulfide groups of the enzyme to sulfhydryl groups, then the precipitated Papain B without added activator should have exhibited approximately the same activity as did the HCN-papain before precipitation. However, the precipitate was found to behave like an unactivatcd papain, since it was completely inactive toward carbobenzoxyisoglutamine and only slightly active toward benzoylarginineamide. The oxidation-reduction theory of papain activation offers no simple explanation for this result. Furthermore, the precipitate, Papain B, on addition of HCK, regained nearly all of the activity of the original HCN-Papain A. This activated Papain B was precipitated a second time by means of isopropyl alcohol, resulting in an inactive enzyme preparation, Papain C, which, on addition of HCN, regained the activity of the original HCX-Papain A. It is difficult to interpret the nearly complete recovery of the original act>ivity after two inactivations and reactivations by HCN in terms of the disulfide-sulfhydryl