Glutathione (GSH, g-glutamylcysteinylglycine), the primary non-protein sulfhydryl in aerobic organisms is synthesized in most cells. The ubiquitous tripeptide is formed by the combination of glutamic acid and cysteine, catalyzed by g-glutamylcysteinyl synthetase. Glycine is then added by glutathione synthetase to form GSH.
In addition to donating an electron during the reduction of hydroperoxides to the respective alcohols (or water in the case of hydrogen peroxide), GSH functions as a co-substrate in the metabolism of xenobiotics catalyzed by glutathione S-transferases. It is also a co-factor for several metabolic enzymes is involved in intracellular transport, functions as an antioxidant and radioprotectant and facilitates protein folding and degradation.
The NWLSS™ Glutathione Assay is a modification of the method first described by Tietze. The general thiol reagent, 5-5'-dithiobis[2-nitrobenzoic acid] (DTNB, Ellman's Reagent) reacts with GSH to form the 412 nm chromophore, 5-thionitrobenzoic acid (TNB) and GS-TNB. The GS-TNB is subsequently reduced by glutathione reductase and β-nicotinamide adenine dinucleotide phosphate (NADPH), releasing a second TMB molecule and recycling the GSH; thus amplifying the response. Any GSSG initially present in the reaction mixture or formed from the mixed disulfide reaction of GSH with GS-TNB is rapidly reduced to GSH and detectable in the assay.