Repairable and Nonrepairable Inactivation of Irradiated Aqueous Papain: Effects of OH, <tex-math>${\rm O}{}_{2}{}^{-},{\rm e}{}_{{\rm aq}}{}^{-}$</tex-math>, and H₂ O₂

Repairable inactivation of papain irradiated in dilute aqueous solutions saturated with air or nitrous oxide is caused predominantly by reversible oxidation of <tex-math>$\text{Cys}_{25}{\rm SH}$</tex-math> by H₂ O₂. The same process occurs in nitrogen-saturated solutions but the yield of repairable product decreases at higher doses, probably because of the consumption of H₂ O₂ by intermediates formed from <tex-math>$e{}_{{\rm aq}}{}^{-}$</tex-math> and papain. The OH radical produces only nonrepairable damage, with the fraction of the OH radical causing nonrepairable inactivation <tex-math>$(f_{{\rm OH}}{}^{{\rm n.r.}})$</tex-math> equal to 0.1 and this is accompanied by, if not solely due to, SH loss. The O₂^- radical with <tex-math>$f_{{\rm O}_{2}}{}^{-{\rm n.r.}}=0.4$</tex-math> also causes nonrepairable damage resulting from or accompanied by SH loss. In addition, there is evidence that every O₂^- reacts with papain to produce a hydrogen peroxide molecule, thus causing a marked increase in the repairable yield. The solvated electron for which <tex-math>$f_{{\rm e}}{}^{{\rm n.r.}}$</tex-math> is 0.07 does not appear to destroy <tex-math>$\text{Cys}_{25}{\rm SH}$</tex-math>, and must, therefore, inactivate papain by damaging other essential residues or changing the active site geometry. The inactivation yields for the present papain solutions prepared by affinity chromatography are compared with other work. Discrepancies in previous determinations of sulfhydryl loss are attributed to the special properties of the sulfenic acid product of the <tex-math>${\rm H}_{2}{\rm O}_{2}\text{-papain}$</tex-math> reaction and its different effects on pHMB and DTNB assays.