GSSG Application

Revision 08/29/06

Overview
Reduced glutathione (GSH) plays a primary role in the protection of the cell against oxidative damage. Two important antioxidant functions include the reduction of hydrogen peroxide (H₂O₂) to water and scavenging free radicals such as the hydroxyl radical (HO^•). An increase in H₂O₂, the presence of other reactive species, or interference with the GSH redox cycle can result in an increase in oxidized glutathione (GSSG).

Figure 1.

GSH Scavenging
4-Vinylpyridine, at a final concentration of 10 mM, was added to a buffer sample containing 1 mM GSH at 18°C. At various time intervals, the GSH was measured by reaction with an excess of 5-5'-dithiobis-2-nitrobenzoic acid (DTNB). The percent remaining GSH was then plotted as a function of time. As shown in Figure 2, 100% of the GSH is removed within 60 minutes.

Calibration
The presence of 4-VP and the reduction in sample dilution requires that the calibrators be treated the same as the test samples. The resulting calibration curve will show significantly reduced reaction rates. As seen in Figure 4 below, the calibration curve maintains linearity.

Figure 2.

• The calibrators are treated the same as the test samples..including MPA extraction.
• 4 N Sodium hydroxide (NaOH) is required but not provided.
• Reagent grade methanol or ethanol.
• 4-Vinylpyridine is required but not provided.
• Extended reaction time

Add 15 mL water to flask
Add 5.2 mL 50% NaOH (19.4 N) to 25 ml volumetric flask
Mix
Cool to room temperature
Bring volume to 25 mL with water Store in a plastic bottle at room temperature

Add 4 g NaOH pellets to 25 ml volumetric flask
Add 20 mL water volumetric flask
Mix until dissolved
Cool to room temperature
Bring volume to 25 mL with water Store in a plastic bottle at room temperature

• 4-VP is typically stored at -20°C and must be brought to ambient temperature before opening the container.
• Add 100 µL 4-VP to a 2-4 mL glass amber vial or other suitable vail with closure.
• Add 1 mL of methanol or ethanol to the vial
• Close vial and store on ice
• The diluted 4-VP should be used on the same day (stability studies of diluted 4-VP are in progress)

1. Prepare a 10 mM solution of GSH in Assay Buffer or equivalent buffer at pH 7-8



Sample	GSH	Buffer	4-VP
Positive Control, µL	100	800	0
Test, µL	100	800	10
Negative Control, µL	0	900	10



2. Stopper tubes and incubate at room temperature for 1 hour.
3. Add 100 µL DTNB Reagent to all tubes.
4. Incubate for 15-30 minutes.
5. Measure the absorbance at 412 nm
6. Calculate the %GSH remaining. (Test-Negative Control)/(Positive Control - Negative Control)

• Prepare a 5% w/v solution of MPA, place on ice.

1. Samples must be immediately placed at 0°C. If the samples cannot be processed immediately, store at -80°
2. Prepare an ice cold 10-20% wet weight/volume homogenate; e.g. 100 mg in 500 µL buffer. The assay requires 200 µL clarified homogenate to measure both GSH and GSSG.
3. Add 150 µL homogenate to a microcentrifuge tube.
4. Centrifuge at 10,000 xg for 2 minutes (or sufficient to clarify the homogenate)
5. Collect supernatant.
6. Add 150 µL supernatant to microcentrifuge tube (save residual for protein determination).
7. Add 150 µL 5% MPA to microcentrifuge tube.
8. Vortex (5 count).
9. Centrifuge at 10,000 xg for 2 minutes (or sufficiently to pellet the precipitate)
10. Place the extact on ice or store at ≤-20°C until assay.
11. Add 200 µL extract to a second microcentrifuge tube (save the residual extract for the GSH determination).
12. Add 10 µL 4 N NaOH to each microcentrifuge vial, mix.
13. Add 10 µL 1 M 4-VP to each microcentrifuge vial, mix.
14. Incubate at room temperature for 1 hour.
15. Proceed with the GSH01 assay.

1. Remove microplate from plastic bag.
2. Bring all reagents to room temperature.
3. Prepare calibrators as per product insert.
4. Add 50 µL of calibrator, diluted sample, and diluted control to a designated well.
5. Add 50 µL of DTNB to wells containing calibrator, control or sample.
6. Add 50 µL of GR to wells containing calibrator, control or sample.
7. Incubate microplate for 3-5 minutes at room temperature.
8. Add 50 µL of reconstituted NADPH to wells containing calibrator, control or sample.
9. Begin recording the absorbance at 405 nm at 15-20 second intervals for 10-15 minutes.
10. Determine the concentration of the controls and samples. If using data reduction on the plate reader, skip steps a-d.Important Note: The Calibrators are supplied as ready-to-use solutions of GSSG. The concentrations assigned to the calibrators are in GSH equivalents or 2 times the GSSG concentration. For the GSSG application, use 1/2 the assigned concentration of the calibrators in the linear regression. See the GSSG example below.
1. Calculate the rate for each calibrator, control and sample from the linear regression of A₄₀₅ as a function of time.
Calculate the linear regression parameters to obtain the equation of the line.
2. Calculate the concentrations of the controls and samples.
3. Correct the control and sample for dilution and report results.

Figure 3.

[GSH]	Rate	Calibration Equation
0	0.005	Slope = 0.0158 Intercept = 0.0041 r2 = 0.9998 Syx = 0.0018 MLD = 0.23
10	0.160
20	0.321
Muscle sample Rate = nmol/mL = nmol/mg wet weight =			0.0295 35.40 0.36

Figure 4.

[GSSG]	Rate	Calibration Equation
0	0.002	Slope = 0.0060 Intercept = 0.0012 r2 = 0.9968 Syx = 0.0016 MLD = 0.52
5	0.029
10	0.062
Muscle sample Rate = nmol/mL = nmol/mg wet weight =			0.0425 6.89 0.0706