Kanazawa H, Fujimoto S, Ohara A
Kyoto Pharmaceutical University, Japan.
Biol Pharm Bull. 1994 Apr;17(4):476-81. doi: 10.1248/bpb.17.476.
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.
在醋酸盐缓冲液（pH 5.6）中，木瓜蛋白酶（EC 3.4.22.2）与抗坏血酸（AsA）和Cu2+一起温育，会通过自由基的位点特异性生成导致酶活性不可逆丧失[H. 金泽、S. 藤本、A. 大原，《生物药物通报》，16，11（1993）]。在本研究中，研究了一些已知的自由基清除剂化合物对Cu(2+)-AsA体系使木瓜蛋白酶失活与AsA氧化之间关系的影响。过氧化氢酶完全保护该酶不被Cu(2+)-AsA体系失活，尽管AsA自氧化过程中已知会产生的过氧化氢（H2O2）本身并不会使该酶失活。过氧化氢酶对AsA的氧化没有影响。硫脲和硫氰酸钠都完全保护该酶不被失活，而AsA仅在初始阶段被部分氧化。在碘化钾存在的情况下，酶的失活和AsA的氧化都具有一个快速的初始阶段，随后是一个无反应发生的稳定阶段，接着是一个较慢的阶段。组氨酸部分阻止了酶的失活和AsA的氧化。目前的结果表明，H2O2作为次级高活性物种的来源，可能是羟基自由基，它是导致失活的原因，并且一些自由基清除剂如硫脲、硫氰酸钠、碘化钾和组氨酸对失活的保护作用是基于在失活的特定位点去除金属离子（Cu2+或Cu+）。
