The mechanism of inactivation of papain (EC 3.4.4.10) by sodium bisulfite was investigated. The inactivation was pH-dependent, and the rate was rapid around pH 6 to 8. Exchange of oxygen for nitrogen gas or addition of radical scavengers largely prevented the inactivation. The results indicate that the inactivating effect of sodium bisulfite is oxygen-dependent and is caused by free radicals formed during the autooxidation of (bi) sulfite. The inactivation was accompanied by a decrease of the essential sulfhydryl group of papain. Treatment of inactivated papain with 2-mercaptoethanol led to partial reactivation with concomitant restoration of the sulfhydryl group. The inactivated papain was judged not to be dimeric on the basis of molecular weight determination. Treatment of papain with 35S-labeled sodium bisulfite resulted in incorporation of a significant amount of radioactivity into the protein. However, the incorporation into iodoacetate-treated papain was slight. Treatment of the labeled protein with 2-mercaptoethanol led to some reduction of the specific radioactivity incorporated with concomitant restoration of the enzyme activity. Based on these results, it is likely that (bi) sulfite inactivates papain through modification or oxidation of the essential sulfhydryl group of the enzyme. In addition, yeast alcohol dehydrogenase (EC 1.1.1.1) was also readily inactivated by (bi) sulfite while lysozyme (EC 3.2.1.17) was resistant to inactivation.
