GSH/GSSG Assay, Cuvette Format

Background

Reduced glutathione (GSH) is a tripeptide (γ-glutamylcysteinylglycine) that contains a free thiol group.).
>Assay Principle

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.) 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.) employed 2 vinylpyridine (2-VP) to derivatize GSH.-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.d84f79-cab6-4e04-99e9-0c09761611fd" height="184" src="/sites/default/files/inline-images/GT40-NSRxn.jpg" width="476" />

Figure 1

: Reaction of the scavanger with a thiol to form a pyridinium salt

Thiol Quantification: The quantitative determination of the total amount of glutathione (GSH + GSSG) employs the enzymatic method first reported by Tietze (1).,5-nitrobenzoic acid (DTNB)) gives rise to a product that can be quantified spectrophotometrically at 412 nm.>

>
References

1.) Analytical Chemistry 27, 502-520.
2.) Analytical Biochemistry 15, 205-210.
3.) Analytical Biochemistry 106, 207-212.
4.) Clinical Chemistry 42, 64-70.
5.) Glutathione in Free Radicals, A Practical Approach, 213.