title: Glutathione and γ-glutamylcysteine in hydrogen peroxide detoxification.
abstract: Hydrogen peroxide (H2O2) is an important regulator of cell redox status and signaling pathways. However, if produced in excess, it can trigger oxidative damage, which can be counteracted by the antioxidant systems. Amongst these, the glutathione (GSH) precursor, γ-glutamylcysteine (γGC), has recently been shown to detoxify H2O2 in a glutathione peroxidase-1 (GPx1)-dependent fashion. To analyze how both γGC and GSH reduce H2O2, we have taken advantage of a colorimetric assay that allows simple and reliable quantification of H2O2 in the micromolar range. Whereas most assays rely on coupled enzymatic reactions, this method determines the formation of a ferric thiocyanate derivative after direct Fe(2+) oxidation by H2O2. Here, we detail the procedure and considerations to determine H2O2 reduction by both γGC and GSH, either from cell samples or in vitro reactions with purified enzymes from GSH metabolism.
authors.affiliation: Institute of Functional Biology and Genomics (IBFG), Department of Biochemistry and Molecular Biology, University of Salamanca-CSIC, Salamanca, Spain.
authors.forename: Ruben
authors.initials: R
authors.lastname: Quintana-Cabrera
authors.forename: Juan P
authors.initials: JP
authors.lastname: Bolaños
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000737:chemistry; D002021:Buffers; D002417:Cattle; D002457:Cell Extracts / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism; D057075:Enzyme Assays / Q000592:standards; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D019001:Nitric Oxide Synthase; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D012015:Reference Standards; D013861:Thiocyanates / Q000737:chemistry; D000094765:Glutathione Peroxidase GPX1
chemical_list: D000975:Antioxidants; D002021:Buffers; D002457:Cell Extracts; D004151:Dipeptides; D016877:Oxidants; D013861:Thiocyanates; C103724:ferric thiocyanate; D006861:Hydrogen Peroxide; D007501:Iron; D005979:Glutathione Peroxidase; D019001:Nitric Oxide Synthase; D005978:Glutathione; C017341:gamma-glutamylcysteine; D000094765:Glutathione Peroxidase GPX1
keywords: Antioxidant; Glutathione; Glutathione peroxidase-1; H(2)O(2); Mitochondria; γ-Glutamylcysteine