Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis

被引:27
作者
Yang, Q
Kratumacher, C
Schilling, D
Pittelkow, MR
Naylor, S
机构
[1] Mayo Clin, Dept Biochem & Mol Biol, Biomed Mass Spectrometry & Funct Proteom Facil, Rochester, MN 55905 USA
[2] Mayo Clin, Dept Dermatol, Rochester, MN 55905 USA
[3] Mayo Clin, Dept Biochem & Mol Biol, Rochester, MN 55905 USA
[4] Mayo Clin, Dept Mol Pharmacol & Expt Therapeut, Div Biomed Engn, Rochester, MN 55905 USA
[5] Mayo Clin, Div Biomed Engn, Clin Pharmacol Unit, Rochester, MN 55905 USA
关键词
D O I
10.1002/bmc.129
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mm ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 muM and a limit of quantitation of 12 muM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples. Copyright (C) 2002 John Wiley Sons, Ltd.
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页码:224 / 228
页数:5
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