Lii C K, Hung C N
Department of Nutrition, Chung Shan Medical College, Taichung, Taiwan, ROC.
Biochim Biophys Acta. 1997 Aug 29;1336(2):147-56. doi: 10.1016/s0304-4165(97)00020-2.
Oxidative stress causes modification of cellular macromolecules and leads to cell damage. The objective of this study was to identify protein modifications that relate to thiol groups in human red blood cells under oxidative stress. With t-butyl hydroperoxide (t-BH) treatment, results of isoelectric focusing (IEF) analysis showed that two dithiothreitol-reversible modifications are observed, one toward the cathode and the other to the anode. Protein change toward the cathode was demonstrated to be hemoglobin oxidation, which gains a net positive charge, based on the same focus on IEF gels as hemoglobin and methemoglobin and molecular weight analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Otherwise, the change toward the anode was the result of mixed disulfide formation between GSH and protein thiols. Based on the results of molecular weight analysis and its reversion from methemoglobin, protein formed mixed disulfides with GSH were also regarded as hemoglobin. As red blood samples were treated with diamide or GSSG, in addition to the mixed disulfides observed in t-BH-treated cells, additional hemoglobin-GSH mixed disulfide appeared. But the disappearance of this diamide-induced additional mixed disulfide by treating cells with t-BH after diamide treatment suggests that the increase of negative charges from GSH are offset by ferrohemoglobin oxidation to ferrihemoglobin. Additionally, other dithiothreitol-reversible modifications of one cell membrane protein, spectrin, were also observed from the formation of high molecular weight molecules as detected by SDS-PAGE. Results indicate that protein thiols in human red blood cells are susceptible to modification under oxidative stress. IEF analysis provides a useful tool to measure methemoglobin and hemoglobin GSH mixed disulfide formation.
氧化应激会导致细胞大分子发生修饰，并引发细胞损伤。本研究的目的是确定在氧化应激下与人类红细胞中硫醇基团相关的蛋白质修饰。用叔丁基过氧化氢（t-BH）处理后，等电聚焦（IEF）分析结果显示观察到两种二硫苏糖醇可逆修饰，一种向阴极移动，另一种向阳极移动。基于在IEF凝胶上与血红蛋白和高铁血红蛋白相同的聚焦情况以及通过SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行的分子量分析，向阴极的蛋白质变化被证明是血红蛋白氧化，其获得了净正电荷。否则，向阳极的变化是谷胱甘肽（GSH）与蛋白质硫醇之间形成混合二硫键的结果。根据分子量分析结果及其从高铁血红蛋白的逆转情况，与GSH形成混合二硫键的蛋白质也被视为血红蛋白。当红细胞样本用二酰胺或氧化型谷胱甘肽（GSSG）处理时，除了在t-BH处理的细胞中观察到的混合二硫键外，还出现了额外的血红蛋白-GSH混合二硫键。但是在二酰胺处理后用t-BH处理细胞，这种二酰胺诱导的额外混合二硫键消失，这表明GSH产生的负电荷增加被亚铁血红蛋白氧化为高铁血红蛋白所抵消。此外，通过SDS-PAGE检测到高分子量分子的形成，还观察到一种细胞膜蛋白血影蛋白的其他二硫苏糖醇可逆修饰。结果表明，人类红细胞中的蛋白质硫醇在氧化应激下易受修饰。IEF分析为测量高铁血红蛋白和血红蛋白GSH混合二硫键的形成提供了一种有用的工具。
