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The Glutathione (total), detection kit is a complete kit that allows the determination of total, reduced, and oxidized glutathione concentrations in your samples. In the first part of the assay the total amount of glutathione (oxidized and reduced forms) is measured. The enzyme Glutathione Reductase provided with the kit reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH. In this way, all glutathione present in the sample will be in reduced form. Subsequently, the chromogen DTNB reacts with the thiol group of GSH to produce a colored compound (TNB) that absorbs at 405 nm. The total glutathione content in unknown samples is determined by comparison with the predetermined glutathione standard curve. The rate of chromophore production is proportional to the concentration of glutathione within the sample. The rate can be determined from the absorbance change over time.
The kit also allows the measurement of GSSG only. For that, samples and standards need to be treated with 4-vinylpyridine. This reagent blocks free thiols present in the sample, thus acting as a GSH quencher. After 4-vinylpyridine treatment, samples are incubated with the reaction mix, which destroys the excess amount of 4-vinylpyridine and converts GSSG to GSH. The rate of color development will therefore be proportional to the concentration of GSSG.
By subtracting the concentration of oxidized glutathione from the concentration of total glutathione, the concentration of reduced glutathione can be determined.