Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

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• 作者：Mario Cappiello (1)
  Eleonora Peroni (1)
  Ambra Lepore (1)
  Roberta Moschini (1)
  Antonella Del Corso (1)
  Francesco Balestri (1)
  Umberto Mura (1)
  
• 关键词：Glutathione ; Gamma ; glutamyltransferase ; Lens ; Glutathione reductase ; Astrocytoma ; Oxidative stress
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2013
• 出版时间：February 2013
• 年：2013
• 卷：405
• 期：5
• 页码：1779-1785
• 全文大小：241KB
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• 作者单位：Mario Cappiello (1)
  Eleonora Peroni (1)
  Ambra Lepore (1)
  Roberta Moschini (1)
  Antonella Del Corso (1)
  Francesco Balestri (1)
  Umberto Mura (1)
  
  1. Department of Biology, Biochemistry Unit, University of Pisa, Via S. Zeno 51, Pisa, 56127, Italy
  
• ISSN：1618-2650

文摘

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG. Fig Colorimetric method for the specific measurement of glutathione. γ-glutamyltransferase (γ-GT) transfers the γ-glutamyl moiety from glutathione to an acceptor (Gly-Gly), with the formation of γ-glutamyl-Gly-Gly and Cys-Gly. The latter dipeptide is hydrolized by leucyl-aminopeptidase (LAP) to form cysteine, which can be easily measured using a colorimetric assay at 560 nm