Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry.
Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry.
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铜，锌超氧化物歧化酶驱动的自由基修饰：铜和碳酸根阴离子引发的蛋白质自由基化学。
DOI：
10.1042/bj20070722
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Mason,RonaldP
中科院分区：
文献类型：
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作者：
Ramirez,DarioC;Gomez-Mejiba,SandraE;Corbett,JeanT;Deterding,LeesaJ;Tomer,KennethB;Mason,RonaldP
野生型SOD1 （Cu， zn -超氧化物歧化酶）与h2o2反应的机制、涉及的氧化剂以及如何产生和产生哪些蛋白质自由基及其命运的了解尚不完整，但需要更好地了解该反应的作用。本研究以人血清白蛋白（HSA）和小鼠脑匀浆（mBH）为靶模型，采用免疫自旋捕获和质谱分析方法研究了人(h)和牛(b) SOD1与h2o2反应时的蛋白氧化。为了获得这一反应的机理信息，我们考虑了铜-和CO3•−（碳酸盐自由基阴离子）启动的蛋白质氧化。我们选择的实验条件可以清楚地将SOD1驱动的CO3•−氧化和由SOD1活性位点释放的铜引发的氧化区分开来。在缺乏（bi）碳酸盐的情况下，SOD1活性位点铜会产生位点特异性自由基介导的断裂。在（双）碳酸盐和DTPA（二乙烯三胺五乙酸）（抑制铜化学）存在的情况下，CO3•−在SOD1和HSA中产生不同的自由基位点，引起蛋白质聚集而不引起蛋白质断裂。hSOD1与h2o2反应产生的CO3•−也在mBH中产生独特的DMPO（5,5-二甲基吡咯啉- n -氧化物）硝基加合物阳性蛋白带。最后，我们提出了一个生化机制来解释二氧化碳产生CO3•−，增强蛋白质自由基形成和（bi）碳酸盐对h2o2诱导的SOD1活性位点断裂的保护。本研究为研究SOD1与H2O2逆反应的分子机理和氧化靶点建立实验条件具有重要意义；这些结果是分析sod1驱动氧化在病理过程（如神经炎症）中的关键目标的第一步。
The understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with H2O2and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and CO3•−(carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO3•−from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), CO3•−produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO3•−produced by the reaction of hSOD1 with H2O2also produced distinctive DMPO (5,5-dimethylpyrroline-N-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO3•−production from CO2, enhanced protein radical formation and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation.