GPx H2O2 + 2GSH -------------> GSSG + 2H2O

GR GSSG + NADPH -------------> 2GSH + NADP+

When cells are exposed to increased levels of oxidative stress, GSSG will accumulate and the ratio of GSH to GSSG will decrease. Therefore, the determination of the GSH/GSSG ratio and the quantification of GSSG are useful indicators of oxidative stress in cells and tissues.

The low concentration of GSSG (high GSH/GSSG ratio) in tissues coupled with the need to prevent GSH oxidation during sample preparation, are important considerations for the accurate measurement of GSSG and GSH/GSSG ratios. Guntherberg and Rost (2) first reported the use of N-ethylmaleimide (NEM) reacting with GSH to form a stable complex, therefore removing the GSH prior to the quantification of GSSG in tissues. Unfortunately, NEM inhibits GR. To overcome this problem, Griffith (3) employed 2-vinylpyridine (2-VP) to derivatize GSH. Although 2-VP does not significantly inhibit GR, it reacts relatively slowly with GSH and is not very soluble in aqueous solutions.

Scavenging of Free Thiols: This assay employs a pyridine derivative as a thiol-scavenging reagent thereby overcoming the shortfalls of both prior methods. At the concentration employed in the assay, this derivative reacts quickly with GSH but does not interfere with the GR activity.

Thiol Quantification: The quantitative determination of the total amount of glutathione (GSH + GSSG) employs the enzymatic method first reported by Tietze (1). Briefly, the reaction of GSH with Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DTNB)) gives rise to a product that can be quantified spectrophotometrically at 412nm. This reaction is used to measure the reduction of GSSG to GSH. The rate of the reaction is proportional to the GSH and GSSG concentration.

G8124-10A: Assay Buffer, 1x100ml. General buffer used to dilute samples and reagents. *G8124-10B: Standard. 1x500ul. 10uM GSSG standard working solution. G8124-10C: Scavenger, 1x3ml. Thiol scavenger to keep GSSG in its oxidized form. G8124-10D: DTNB, 1x1vial. Lyophilized 5,5'-dithiobis-2nitrobenzoic acid G8124-10E: MPA 5%, 1x20ml. 5% metaphosphoric acid solution used to deproteinate samples *G8124-10F: Reductase, 1x35ul. Recombinant glutathione reductase. *G8124-10G: NADPH, 1x1vial. Lyophilized beta-nicotinamide adenine dinucleotide phosphate

Store *G8124-10B, *G8124-10F and *G8124-10G at -20°C. Store other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.