Protein thiol modifications of human red blood cells treated with t-butyl hydroperoxide.
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.
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Department of Nutrition, Chung Shan Medical College, Taichung, Taiwan, ROC. hawwen@mercury.csmc.edu.tw
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D000328:Adult; D001798:Blood Proteins / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D018384:Oxidative Stress; D010545:Peroxides / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D020122:tert-Butylhydroperoxide
D001798:Blood Proteins; D004220:Disulfides; D010545:Peroxides; D013438:Sulfhydryl Compounds; D020122:tert-Butylhydroperoxide; D005978:Glutathione