Glutathione Reductase, Colorimetric Kit, BioAssay™

Glutathione reductase (GR, EC 1.6.4.2) is a ubiquitous enzyme, which catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH). Glutathione reductase is essential for the glutathione redox cycle that maintains adequate levels of reduced cellular GSH. GSH serves as an antioxidant, reacting with free radicals and organic peroxides, in amino acid transport, and as a substrate for the glutathione peroxidases and glutathione Stransferases in the detoxification of organic peroxides and metabolism of xenobiotics, respectively.1||This homodimeric enzyme is a member of the family of flavoprotein disulfide oxidoreductases. Each subunit has four domains; beginning at the N-terminus: an FAD-binding domain, an NADPH-binding domain, a central domain, and an interface domain. The active site of GR is at the dimeric interface. Since the GSSG binding site is composed of residues from both subunits, only the dimeric form is active.2|| Glutathione Reductase|GSSG + NADPH + H+ -----------------------------------> 2GSH + NADP+||Oxidized glutathione is reduced by a multi-step reaction in which GR is initially reduced by NADPH forming a semiquinone of FAD, a sulfur radical and a thiol. The reduced GR (GRred) reacts with a molecule of GSSG, resulting in a disulfide interchange, which produces a molecule of GSH and the GRred-SG complex. An electron rearrangement in GRred-SG results in a second disulfide interchange, splitting off the second molecule of GSH and restoring the GR to the oxidized form.3 Table1 lists some properties of glutathione reductase.||Principle of the Assay:|This assay is based on the oxidation of NADPH to NADP+ catalyzed by a limiting concentration of glutathione reductase. One GR activity unit is defined as the amount of enzyme catalyzing the reduction of one micromole of GSSG per minute at pH 7.6 and 25°C. As shown in the above reaction, one molecule of NADPH is consumed for each molecule of GSSG reduced. Therefore, the reduction of GSSG is determined indirectly by the measurement of the consumption of NADPH, as demonstrated by a decrease in absorbance at 340nm (A340) as a function of time.||Reagents:|1. NADPH: Lyophilyzed solution containing NADPH, Tris, manitol. 6 vials|2. GSSG: Oxidized Glutathione in K•PO4. Buffer containing EDTA. pH 7.5|3. K•PO4: Potassium Phosphate Buffer, pH 7.5, 20ml|4. Diluent Buffer: Potassium Buffer containing BSA/EDTA