Offer T, Mohsen M, Samuni A
Department of Molecular Biology, Faculty of Medicine, The Hebrew University, Jerusalem, Israel.
Free Radic Biol Med. 1998 Nov 1;25(7):832-8. doi: 10.1016/s0891-5849(98)00162-2.
Nitroxide stable free radicals have previously been found to afford protection in various biological systems against diverse types of oxidative stress, including, ischemia/reperfusion, hyperoxia, mechanical trauma, toxic xenobiotics, ionizing radiation, gastric and colonic irritants or strong oxidants. Dismutation of superoxide has originally been suggested to be one of the mechanisms that underlie the anti-oxidant effect of nitroxides. However, no direct evidence has been found, so far, to support this assumption. In the present study, superoxide and H2O2, generated enzymatically, were used to directly inactivate papain, a sulfhydryl enzyme, in vitro. The rate of papain inactivation served to assess the damage. The reaction mixtures contained a chelate in order to prevent the effect of adventitious redox-active metal ions, pre-empt the Fenton reaction and avoid hydroxyl-induced damage. Catalase or SOD alone partially protected the papain from inactivation. The protective effect of nitroxides resembled that of SOD in several aspects: a) nitroxides provided partial protection; b) the protective effect of nitroxides did not increase with the elevation of their concentration (above 0.5 mM); c) the combined addition of SOD and the nitroxide did not provide greater protection than that demonstrated by nitroxides or SOD separately; d) the effects of catalase with the nitroxide were additive; e) the nitroxide, like SOD itself, did not protect papain from H2O2-induced inactivation; f) the nitroxide was found not to be consumed in the course of the reaction but rather to be recycled. The results indicate that: (a) the main species responsible for the papain inactivation in a system in which the effect of transition metals is pre-empted, are O2-. and H2O2; (b) nitroxides inhibit the oxidative damage by removing superoxide not stoichiometrically, but rather catalytically as SOD-mimics; (c) nitroxides do not afford protection when the oxidative damage is induced directly by H2O2 (and not mediated by redox-active metals).
此前已发现氮氧化物稳定自由基可在各种生物系统中对多种类型的氧化应激提供保护，这些氧化应激包括缺血/再灌注、高氧、机械创伤、有毒外源性物质、电离辐射、胃和结肠刺激物或强氧化剂。超氧化物歧化最初被认为是氮氧化物抗氧化作用的潜在机制之一。然而，迄今为止尚未找到支持这一假设的直接证据。在本研究中，通过酶促产生的超氧化物和过氧化氢被用于在体外直接使木瓜蛋白酶（一种巯基酶）失活。木瓜蛋白酶失活的速率用于评估损伤情况。反应混合物中含有一种螯合剂，以防止偶然的氧化还原活性金属离子的影响，预先阻止芬顿反应并避免羟基诱导的损伤。单独的过氧化氢酶或超氧化物歧化酶可部分保护木瓜蛋白酶不被失活。氮氧化物的保护作用在几个方面类似于超氧化物歧化酶：a）氮氧化物提供部分保护；b）氮氧化物的保护作用不会随着其浓度升高（高于0.5 mM）而增强；c）超氧化物歧化酶和氮氧化物联合添加所提供的保护并不比单独使用氮氧化物或超氧化物歧化酶时更强；d）过氧化氢酶与氮氧化物的作用是相加的；e）氮氧化物与超氧化物歧化酶本身一样，不能保护木瓜蛋白酶免受过氧化氢诱导的失活；f）发现氮氧化物在反应过程中不会被消耗，而是会被循环利用。结果表明：（a）在预先排除过渡金属影响的系统中，导致木瓜蛋白酶失活的主要物质是超氧阴离子和过氧化氢；（b）氮氧化物通过非化学计量地去除超氧化物，而是作为超氧化物歧化酶模拟物进行催化作用来抑制氧化损伤；（c）当氧化损伤直接由过氧化氢诱导（而非由氧化还原活性金属介导）时，氮氧化物不提供保护作用。
