Effect of Radical Scavengers on the Inactivation of Papain by Ascorbic Acid in the Presence of Cupric Ions

Abstract

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu²⁺ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm. Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu²⁺ -AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu²⁺ -AsA system, although hydrogen peroxide (H₂O₂) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H₂O₂ serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu²⁺ or Cu⁺) at the specific site of inactivation.