Total Glutathione assay kit

Total Glutathione assay kit

Product Description

Glutathione (GSH) is the most abundant thiol compound in animal tissues, plant tissues, bacteria, and yeast. GSH has many different roles, including protection against reactive oxygen species and the maintenance of protein thiol groups. During these processes, GSH is converted into its oxidized form, glutathione disulfide (GSSG). Since GSSG is then enzymatically reduced by glutathione reductase, GSH is the dominant form in organisms. DTNB (5,5 EDithiobis(2-nitrobenzoic acid)), known as Ellman’s Reagent, was developed to detect thiol compounds. In 1985, Dr. M. E. Anderson suggested that the glutathione recycling system involving DTNB and glutathione reductase could be used as a highly sensitive glutathione detection method.

DTNB and GSH react to generate 5-Mercapto-2-nitrobenzoic acid (TNB) in Fig. 1. Since TNB is yellow, the GSH concentration in a sample solution can be determined by O.D. measurement at 412 nm absorbance (Fig. 2). GSH is regenerated from GS-TNB by glutathione reductase and will again react with DTNB to produce TNB. This recycling reaction improves the sensitivity of total glutathione detection. Total Glutathione Quantification Kit contains all of the necessary reagents for total glutathione measurement, except for those used in sample preparation. 5-Sulfosalicylic acid is recommended for the removal of proteins from sample solutions and for the prevention of GSH oxidation and γ-glutamyl transpeptidase reactions. However, the optimum method for sample preparation differs from sample to sample, so please review the references. This kit can be used to quantify total glutathione concentrations from 1 μM to 100 μM using the standard method. For lower glutathione concentrations, such as in blood samples, longer incubation times are required.

Performance characteristics

• Assay type: colorimetric detection kit
• Sample types: whole blood, serum, plasma, erythrocytes, urine, cell lysates, and tissue samples
• Sensitivity: 0.634 uM
• Standard curve range: 0.78 uM–25 uM
• Reactivity: species independent

Background

Glutathione (GSH) is the highest concentration non-protein thiol in mammalian cells and is present in concentrations of 0.5–10 mM. GSH plays a key role in many biological processes, including the synthesis of proteins and DNA, the transport of amino acids, and the protection of cells against oxidation. Harmful hydrogen peroxide cellular levels are minimized by the enzyme glutathione peroxidase (GP) using GSH as a reductant. The oxidized GSH dimer, GSSG, is formed from GSH and peroxide by the GP reaction. An important role of GSSG in the NFkB activating signal cascade is suggested by the facts that the potent NFk B inducer, tetradecanoyl phorbol acetate, increases intracellular GSSG levels and GSSG/GSH ratios. Glutathione S-transferases (GST) are an important group of enzymes that catalyze the nucleophilic addition of GSH to electrophiles. They are encoded by 5 gene families; 4 encode cytosolic GST and one encodes the microsomal form of GST.

Assay principle

The Glutathione Colorimetric Detection Kit is designed to quantitatively measure glutathione (GSH), and oxidized glutathione (GSSG) present in a variety of samples. No separation or washing is required. A GSSG standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The kit utilizes a colorimetric substrate that reacts with the free thiol group on GSH to yield a highly colored product. Supplied reagents are in solution and require simple dilution for use in the assay. By using 2-vinylpyridine (not supplied) to block any free GSH in the sample, oxidized glutathione (GSSG) can be determined. Any samples that have not been treated with 2-vinylpyridine will yield Total GSH levels. The Free GSH concentration in the sample is calculated from the difference between the Total GSH determined and the GSH generated from oxidized glutathione for the 2-vinylpyridine-treated samples. The concentration of GSH can be determined either as an endpoint read of the color developed at 405 nm or by measuring the rate of color development at 405 nm.