Solute Radical Inactivation of the Sulfhydryl Enzyme Papain – Sensitization and Protection

Abstract

Particularly pure N2O-purged papain solutions (2 × 10−5 M; pH 6.8) were irradiated at 0 °C for different times with 60Co γ-rays, in the presence of 10−30 mM solutions of acetate, isobutanol, and thiocyanate. In comparison to OH radicals, these solute radicals substantially enhanced papain inactivation. Calculated inactivation probabilities per papain–radical reaction (i.e. 0.43 for OH and 1.00 for solute radicals) support this finding.In situ protection of both N2-purged and N2O saturated papain solutions by cysteine was found to occur only at cysteine: papain molar ratios above 15:1, while below this ratio substantial sensitization occurred, reaching a maximum at a 2:1 ratio. This enhanced inactivation of papain at low cysteine concentrations is primarily repairable in nature, and probably due to CysS• attack on papain's active site sulfhydryl. Only at relatively high cysteine:papain ratios (> 30) can protection be ascribed simply to radical scavenging.