On the mechanism of inactivation of active papain by ascorbic acid in the presence of cupric ions.
An inactivation mechanism of active papain (EC 3.4.22.2) by the Cu(2+)-ascorbic acid (AsA) system was examined. Incubation of active papain, which contains an active sulfhydryl (SH) group, with the Cu(2+)-AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. The enzyme was not inactivated at a molar ratio of enzyme to Cu2+ of 1:< 1, whereas at a molar ratio of 1:1-2, the extent of inactivation showed the same dependence on the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. The degree of inactivation was dependent on the decrease in SH content of the enzyme. Catalase at a low concentration partially protected the enzyme from inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and AsA from oxidation. The present results suggest that an additional function of H2O2, besides producing hydroxyl radicals (.OH), is to promote the conversion of Cu+ into Cu2+, and that an active SH group of papain is site-specifically modified by the .OH, resulting in inactivation of the enzyme.
Kyoto Pharmaceutical University, Japan.
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Kanazawa
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Fujimoto
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Ohara
D000596:Amino Acids / Q000032:analysis; D001205:Ascorbic Acid / Q000494:pharmacology*; D001586:Benzoylarginine Nitroanilide / Q000737:chemistry; D001665:Binding Sites; D002374:Catalase / Q000378:metabolism; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D016166:Free Radical Scavengers; D006639:Histidine / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010084:Oxidation-Reduction; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000596:Amino Acids; D016166:Free Radical Scavengers; D013438:Sulfhydryl Compounds; D006639:Histidine; D003300:Copper; D001586:Benzoylarginine Nitroanilide; D006861:Hydrogen Peroxide; D002374:Catalase; D010206:Papain; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid