Summary

Glutathione Reductase is a ubiquitous 100-120 kD dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using b-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor. A measurable fraction of GR derived from various sources is often found as the inactive apoenzyme. Dietary supplementation of riboflavin or thyroxin has been shown to activate GR. The in vitro addition of FAD is the basis of assessing riboflavin deficiency. The active site for GR contains a flavin adenine dinucleotide (FAD) and a disulfide. In the presence of NADPH, there is a two electron reduction of GR to produce a semiquinone of FAD, a sulfur radical and a thiol. Purified GR tends to form aggregates in the absence of thiols and these aggregates retain full enzymatic activity. Purified enzyme is reversibly inactivated by NADPH, b-nicotinamide adenine dinucleotide (NADH), GSH, dithionite or borohydride. This inactivation likely requires the presence of divalent cations such as Zn++ and Cd++. The GR enzyme is fully protected from inactivation by ethylenediamine tetraacetic acid (EDTA). Inactivated GR is activated by GSSG, NADP+ and NAD+. In vivo, GR activity is regulated through a redox interconversion mechanism mediated by GSSG regulation of the NADPH generating pathways.

The NWLSS™ Glutathione Reductase Activity Assay is based on monitoring the oxidation of NADPH as a decrease in absorbance at 340 nm as shown below.

For each mole of GSSG reduced, one mole of NADPH is oxidized resulting in loss of absorbance at 340 nm. One Unit GR activity is defined as the amount of enzyme that will reduce 1 mmole GSSG per minute at pH 7.6 and 25C.