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GSH Assay Protocols

Glutathione or GSH is a protein found in both plants and animals. It works to help enzyme reactions in the detoxification of environmental contaminants. In addition, it facilitates amino acid transport within cells and helps maintain protein sulfhydryl reduction reactions. GSH Assay protocols use GSH buffers, standards, substrate, enzymes and reagents. Following the specific procedure is pertinent to the outcome of the assay.

Enzymatic Recycling Method
- The enzymatic recycling method, for GSH, uses glutathione reductase to quantify GSH. This protocol is specifically for plasma, tissue or cultured cell samples. Add the reagent DTNB (5-5'-dithio-bis-2-nitrobenzoic acid) to GSH. The sulfhydryl group in GSH reacts with the reagent and produces a yellow acid called 5-thio-2-nitrobenzoic acid (TNB). Add glutathione reductase to the acid and GSH mixture to recycle GSH and produce more acid. In this procedure, the rate of acid production is proportional to the recycling reaction of GSH; therefore, measure the absorbance of the TNB acid using a spectrophotometer. The absorbance provides an estimate of the actual amount of GSH in the sample.
Liver Cell Glutathione Assay
- This protocol analyzes the amount of GSH in human hepatocarcinoma (liver cancer) cells. To conduct this procedure, use the colorimetric method for GSH determination to reduce and oxidize GSH. Reduce GSH using 5-5'-dithiobis (2-nitrobenzoic acid or DTNB) to form the
  
  colored product 2-nitro-5-thiobenzoic acid and oxidized glutathione (GSSG). Use glutathione reductase to reduce GSSG to form GSH again. In order to measure the amount of GSH in the liver cells, read the sample at 415 absorbance kinetically on a microplate reader.
Cell Apoptosis
- In this protocol, GSH is used to detect cell apoptosis (death) through the measurement of the decrease in reduced GSH. A decrease in the concentration of cellular GSH is an indicator of cell death. Stain the sample cells with non-fluorescent thiolite green. The thiolite green accumulates in the cytosol (cell plasma) and mitochondria (powerhouse) of the cell. Use a flow cytometer at 490/520 nanometers to visualize the amount of GSH in the cells.
Total Concentration of GSH in Animal and Plant Samples
- Measure the concentration of total GSH in both the reduced and oxidized form in different animal and plant samples. Add the general thiol reagent, 5-5'-dithiobis[2-nitrobenzoic acid]. The thiol reagent reacts with GSH to form 412 nm chromophore, 5-thionitrobenzoic acid (TNB) and GS-TNB. Add glutathione reductase and Beta-nicotinamide adenine dinucleotide phosphate (NADPH) to reduce GS-TNB. The glutathione will also release a second TMB molecule and recycle the GSH. In addition, any oxidized GSH (GSSG) present in the mixture fromed from the disulfide reaction is also reduced to GSH. Use a microplate reader at 405 nanometers to read the absorbance level and indicate the total concentration of GSH.

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