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. The included glutathione standards or treated samples are added to the microtiter plate wells, followed by DTNB and glutathione reductase. Addition of beta-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, followed over a 5 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 (see figure below). 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. The series of reactions involved in this assay are illustrated in Figure 1 below.

By adapting the above assay process into a 96 well microtiter plate format, it is now possible to readily measure total GSH (tGSH) in a large number of samples at one time.

G8135-29A: Glutathione Standard, 2mM (reduced form GSH), 1 x 100ul Storage: -20°C

G8135-29B: 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), 1 x 1 vial Supplied as a lyophilized powder. Storage: Room temperature

G8135-29C: beta-Nicotinamide adenine dinucleotide phosphate, reduced form (b-NADPH2), 1 x 1 vial Supplied as a lyophilized powder. Storage: -20°C

G8135-29D: Glutathione Oxidoreductase, 1 x 35ul Storage: 4°C

G8135-29E: Assay Buffer, 1 x 100ml Storage: 4°C

G8135-29F: Metaphosphoric acid (MPA), 5%, 1 x 60ml Storage: 4°C

G8135-29G: Microtiter Plate, 1 x 96wells Storage: Room temperature

Note: The materials provided in this kit are sufficient for at least 96 assays, 40 samples in duplicate plus the standards.

Store the kit components as specified in the Kit Components section. Kit must be used within 6 months of purchase.

1. Add 50ul of each GSH standard or diluted or undiluted sample extract to each well of the microtiter plate. Both standards and samples are analyzed in duplicate or triplicate as needed.

2. Add 50ul each of prepared DTNB and Glutathione Oxidoreductase to each well

3. Incubate the plate for 5-10 minutes at room temperature.

4. Add 50ul of the prepared beta-NADPH2 solution to each well to start the reaction.

5. Within 1 minute after the addition of beta-NADPH2, read the plate using a kinetic program which can monitor the reaction at 1 minute intervals for 4 minutes. The rate of color change is monitored in the 405-412nm (depending on the wavelengths of filters available on your plate reader). Note: If your plate reader does not have a kinetic reading function, read the plate and record the OD value at 0 minute and again at 10 minutes.

Plot the standard curve with Mean vs. GSH concentrations. The total GSH concentration of the samples can be calculated from the standard curve.

If a kinetic microplate reader is not available, the Mean V (the rate of color change) can be estimated by subtracting the OD value at 0 minute from the OD value at 10 minutes and dividing the result by 10. That is,

Mean V = (OD at 10 minutes - OD at 0 minute) / 10

The rate of color change represented as Mean V is proportional to the GSH standard concentrations.