Physiological values for the concentration of intracellular GSH generally range from 1 to 10 mM. Although several methods have been described for the assay of GSH, most of the reliable ones have been labor intensive.
This kit employs a kinetic enzymatic recycling assay, based on the oxidation of GSH by 5,5’-dithiobis-(2-nitrobenzoic acid), [DTNB] to measure the total glutathione (tGSH) content of biological samples. Glutathione standards or treated samples are added to the microtiter plate wells, followed by DTNB and glutathione reductase. Addition of NADPH2 to the wells initiates the progressive reduction of DTNB by GSH, causing a color increase that is monitored at 405 nm. The rate of color change, typically followed over a 4-minute time period, is proportional to the tGSH concentration. Consequently, the concentration of tGSH in unknown samples may be determined by reference to the standard curve. GSH reacts with DTNB to produce a colored ion, which absorbs light at 405 nm, and a mixed disulphide. This disulphide reacts with further quantities of GSH present to liberate another ion and GSSG. GSSG is reduced enzymatically to GSH which then re-enters the cycle. Since GSSG represents only a small percentage of total acid-solution free glutathione, the resulting values for tGSH (which encompasses both GSH and GSSG) are expressed in units of GSH equivalents. Instructions are provided for analysis of tGSH in a wide range of samples, including tissue extracts, cell lysates, blood, urine, and saliva.