氧自由基对CAT、SOD和GPX的氧化修饰研究

Effects of Oxygen Free Radicals on Functions and Structures of CAT, SOD and GPX in vitro

以氧自由基产生系统作用于CAT、SOD和GPX,探索氧自由基的氧化修饰对CAT、SOD和GPX结构与功能的影响。结果:CAT被氧自由基氧化修饰后,改变了酶蛋白二级结构(α-螺旋百分含量减少)、空间构象(ΔCp减少,分子粒晶直径增大)、辅基Fe(Ⅲ)微环境及酶、底物的亲和力和催化活性。过氧化氢 羟自由基系统可以使SOD酶蛋白氨基酸残基氧化产生羰基,并使SOD活性降低。过氧化氢 羟自由基可在His对SOD作用背景上更为严重地损伤SOD(活性降低幅度更大)。氧自由基对GPX有损伤作用:氧自由基可使GPX酶蛋白氨基酸残基氧化产生羰基,并可使GPX酶蛋白二级结构发生变化(α-螺旋百分含量减少),这些可能是使GPX活性下降的原因。结果提示:氧自由基的氧化修饰,可对抗氧化酶CAT、SOD和GPX造成损伤。氧自由基使酶蛋白结构和辅基的微环境与价态发生变化,可能是酶活性降低的原因。

Exposure of enzyme proteins to oxidants leads to increased oxidation followed by changes of function and structure The role of superoxide anions, hydrogen peroxides and/or hydroxyl free radicals in oxidative modification of superoxide dismutase(SOD), catalase(CAT) and glutathione peroxidase(GPX)and changed structures and decreased activities of these proteins in vitro was examined in this study Subsequent to treatment of these proteins, CAT, SOD and GPX, with superoxide aniongenerating system, these proteins were exposed to hydrogen peroxide or xanthine/xanthine oxidase, there were decreased αhelix structures and decreased activities These changes in protein structure and activity were similar with the superoxide anions , hydrogen peroxides and/or hydroxyl free radicals About oxidized CAT by superoxide anions, the remnant activity of the sample was about 56% of native enzyme; the apparent Km value increased from 4716?mmol/L to 8986?mmol/L, that means the binding ability of CAT with substrate was decreased; in CD spectra, the αhelix decreased from 2320% to 1810% ; the microenvironment of prosthetic group Fe(III) changed, in ESR spectra ΔHpp decreased from 6445?×10-4T to 2930?×10-4T and curve pattern changed from Lorentz to Gauss; in DSC spectra inflection point temperature was from 5777?℃ to 5008?℃, and ΔCp was from 0272 W/g to 0207 W/g; in SAXS spectra molecular granule diameter and distribution changed from 105 (9592%),245?(212%),279?(196%) to 103?(6261%),286?(3242%),345?(497%); carbonyl groups in the proteins were formed, it was 0357?μmol/L(/mg protein) after damaged 20 minutes These results showed that the oxidation of superoxide anions on CAT protein and the changes of prosthetic group Fe(III) microenvironments may be main mechanism of the activity change of the oxidized CATAbout oxidized SOD by hydrogen peroxide and/or hydroxyl free radicals, the remnant activity of the sample was about 45% of native enzyme; carbonyl groups in the proteins were formed, it was 0594?μmol/L(/mg protein) after damaged 20 minutes; the damage was more serious with Histidine, the remnant activity of the sample was about 165% of native enzyme And about oxidized GPX by superoxide anions, same as above, the remnant activity of the sample was about 65% of native enzyme; carbonyl groups in the proteins were formed, it was 0254?μmol/L(/mg protein) after damaged 20 minutes; and also the secondary structure was changed These results showed that the oxidation of superoxide anions on SOD and GPX protein may be main mechanism of the activity change of the oxidized SOD and GPX