Analysis of GSH and GSSG after derivatization with N-ethylmaleimide
Analysis of GSH and GSSG after derivatization with N-ethylmaleimide
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DOI：
10.1038/nprot.2013.095
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发表时间：
2013-09-01
期刊：
影响因子：
14.8
通讯作者：
Rossi, Ranieri
中科院分区：
文献类型：
--
作者：
Giustarini, Daniela;Dalle-Donne, Isabella;Rossi, Ranieri
本方案描述了测定血液和其他组织中谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）浓度的程序。在用任何常用的酸对生物样品进行脱蛋白的过程中，样品中发现的5-15%的GSH可能被人为氧化为GSSG，从而显著高估GSSG。这可以通过在酸脱蛋白之前用烷基化剂N-乙基马来酰亚胺（NEM）衍生GSH以形成GS-NEM，然后用二氯甲烷从脱蛋白样品中反萃取过量NEM来防止。然后，基于谷胱甘肽还原酶和NADPH将GSSG转化为GSH并与5，5 '-二硫代双-（2-硝基苯甲酸）反应，采用GSH再循环方法通过分光光度法测量GSSG浓度。相反，通过以下两种方法中的任一种测量GSH浓度：通过HPLC分析用于测量GSSG的相同样品中的GS-NEM结合物，或者通过分光光度法（GSH再循环法）分析未用NEM衍生的另一份样品等分试样中的GSH。该方法可以在30分钟或更短的时间内测定血液和其他组织中的GSH和GSSG。
This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.