Glutathione (gamma-glutamylcysteinylglycine or GSH) is a naturally occuring tripeptide whose nucleophilic and reducing properties play a central role in metabolic pathways, as well as in the antioxidant system of most aerobic cells.1 GSH plays a critical role as a coenzyme with a variety of enzymes including, glutathione peroxidase, glutathione S-transferase and thiol transferase. GSH also plays major roles in drug metabolism, calcium metabolism, the gamma-glutamyl cycle, blood platelet and membrane functions. In addition, GSH is crucial to a variety of life processes, including the detoxification of xenobiotics, maintenance of the -SH level of proteins, thiol-disulfide exchange, removal of hydroperoxides and free radicals, and amino acid transport across membranes. Physiological values of intracellular GSH generally range from 1 to 10mM. Although many methods have been described for the assay of GSH, the reliable ones are labor intensive and not easy to use.
Principle of the Assay:
This colorimetric method for GSH quantitation is based on a chemical reaction, which proceeds in two steps. The first step leads to the formation of substitution products (thioethers) between a patented reagent, R1 (4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate), and all mercaptans (RSH) which are present in the sample.
The second step is a beta-elimination reaction which takes place under alkaline conditions. This reaction is mediated by reagent R2 (30% NaOH) which specifically transforms the substitution product (thioether) obtained with GSH into a chromophoric thione which has a maximal absorbance wavelength at 400nm.
This method makes it possible to specifically assay glutathione with only one sampling and one colorimetric measurement. A modification of this method can be used to assay other mercaptans. This is based on the measurement of substitution products (thioethers) which absorb light at 356nm in the absence of reagent R2.
Because of its simplicity, this method is especially well adapted to the assay of glutathione in large series of biological samples. The main advantage of the method is that it is specific for glutathione and it does not require use of an enzyme as a reagent.
1. G8123-50A: Reagent (R1) 1x5.5ml
2. G8123-50B: 30% NaOH (R2) 1x10ml
3. G8123-50C: Buffer (solution 3) 1x100ml
These solutions are ready for use.
Storage and Stability:
Reagent bottles should always be kept tightly closed and stored at 4 degrees C. For each experiment, take out the required amount of buffer and reagents. Transfer should be made using clean pipette tips to avoid contamination. Immediately close the remaining buffer and reagent bottles and store them at 4 degrees C. Do not leave reagent bottles open on the bench, at room temperature or exposed to light. Under the above conditions, all reagents are stable for 12 months.