Effect of radical scavengers on the inactivation of papain by ascorbic acid in the presence of cupric ions.
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 Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-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.
Kanazawa
H
H
Kyoto Pharmaceutical University, Japan.
Fujimoto
S
S
Ohara
A
A
D001205:Ascorbic Acid / Q000494:pharmacology*; D001586:Benzoylarginine Nitroanilide / Q000737:chemistry; D002374:Catalase / Q000494:pharmacology; D016166:Free Radical Scavengers; D006639:Histidine / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009942:Organometallic Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010206:Papain / Q000037:antagonists & inhibitors*; D011193:Potassium Iodide / Q000494:pharmacology; D017382:Reactive Oxygen Species; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013861:Thiocyanates / Q000494:pharmacology; D013890:Thiourea / Q000494:pharmacology
D016166:Free Radical Scavengers; D009942:Organometallic Compounds; D017382:Reactive Oxygen Species; D013861:Thiocyanates; D011193:Potassium Iodide; C015092:cupric acetate; D006639:Histidine; C024553:sodium thiocyanate; D001586:Benzoylarginine Nitroanilide; D006861:Hydrogen Peroxide; D002374:Catalase; D010206:Papain; D013890:Thiourea; D001205:Ascorbic Acid