Identification of proteins containing H2O2-sensitive cysteine residues

开发了一种用于检测含有H2O2敏感的半胱氨酸（或硒代半胱氨酸）残基的蛋白质的方法，以研究细胞中H2O2对蛋白质的氧化。该程序是基于这样的事实，即H2O2和生物素偶联碘乙酰胺（BIAM）选择性和竞争性地与表现出低pKa的半胱氨酸残基反应，并且可以通过链霉亲和素印迹分析来监测由细胞先前暴露于H2O2或诱导H2O2产生的试剂引起的BIAM标记细胞裂解物蛋白的减少。这个程序被应用到大鼠嗜铬细胞瘤PC12细胞直接处理过氧化氢，小鼠海马HT22细胞，其中H2O2的生产是由谷氨酸诱导，和人红白血病K562细胞，其中H2O2的生产是由佛波醇肉豆蔻酸酯乙酸诱导，它揭示了几种细胞蛋白质含有半胱氨酸或硒代半胱氨酸残基，选择性氧化过氧化氢。这些H2O2敏感的蛋白质被确定为蛋白质二硫键异构酶家族，硫氧还蛋白还原酶和肌酸激酶，所有这些都是以前已知的，在其催化位点含有至少一个反应性半胱氨酸或硒代半胱氨酸的成员。因此，该程序应证明是有用的蛋白质的识别，被氧化的H2O2响应于各种细胞外试剂产生。

A procedure for detecting proteins that contain H2O2-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H2O2 in cells. The procedure is based on the facts that H2O2 and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a low pKa, and that the decrease in the labeling of cell lysate proteins with BIAM caused by prior exposure of cells to H2O2 or to an agent that induces H2O2 production can be monitored by streptavidin blot analysis. This procedure was applied to rat pheochromocytoma PC12 cells directly treated with H2O2, mouse hippocampal HT22 cells in which H2O2 production was induced by glutamate, and human erythroleukemia K562 cells in which H2O2 production was induced by phorbol myristate acetate, and it revealed that several cell proteins contain cysteine or selenocysteine residues that are selectively oxidized by H2O2. Three of these H2O2-sensitive proteins were identified as a member of the protein disulfide isomerase family, thioredoxin reductase, and creatine kinase, all of which were previously known to contain at least one reactive cysteine or selenocysteine at their catalytic sites. This procedure should thus prove useful for the identification of proteins that are oxidized by H2O2 generated in response to a variety of extracellular agents.