13/04/03

Oxidized Glutathione (GSSG Application)

Home | About Us | Products | Technical Support | Links | Contact
Us/Place an Order"Premier Products for Superior Life Science Research"GSSG Application
Revision 05/17/12

Overview
Reduced glutathione (GSH) plays a primaryrole in the protection of the cell against
oxidative damage. Two important antioxidant
functions include the reduction of hydrogenperoxide (H2 O 2 ) to water and scavenging
free radicals such as the hydroxyl radical(HO• ). An increase in H2 O 2 , the presence ofother reactive species, or interference withthe GSH redox cycle can result in an
increase in oxidized glutathione (GSSG).
Figure 1.
The NWLSS™GSH01 Assay standard method measures "total" glutathione (GSHt). The GSHt in thesample is defined as the concentrations of GSH and GSSG (The mixed disulfides of GSH and cysteine orproteins are not included in the definition). However, it is also possible to measure the GSSG
concentration of the sample by first treating the sample with 4-vinylpyridine (4-VP) to sequester GSH.
The estimated GSSG concentration is highly influenced by sample history and treatment. Ex vivo, GSSG
concentration can increase due to continued reduction of hydrogen peroxide by glutathione peroxidase
and reaction with other reactive species. This is especially true for cells that have been lysed, evenwhen refrigerated. C onversely, the GSSG concentration in the sample can be under estimated if theglutathione redox cycle is active or by mixed disulfide reactions with sulfhydryls other than GSH. Theseissues can be minimized by maintaining the sample at 0°C (ice bath) and extracting withmetaphosphoric acid as soon as possible. Once extracted, the protein free acidic sample can be stored
at -20°C for several months or indefinitely at -80°C .

GSH Scavenging
4-Vinylpyridine was added to a buffer sample
containing 1 mM GSH at 18°C (finalconcentration 4VP is approximately 10 mM).
At various time intervals, the GSH wasmeasured by reaction with an excess of 5-5'dithiobis-2-nitrobenzoic acid (DTNB). The
percent remaining GSH was then plotted as afunction 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 insample dilution requires that the calibrators
be treated the same as the test samples. The
resulting calibration curve will show
significantly reduced reaction rates. As seenin Figure 4 below, the calibration curve
nwlss.com/products/pdf/gsh01gssgapp051712.htm

Figure 2.
1/3

13/04/03

Oxidized Glutathione (GSSG Application)

maintains linearity.

Procedure
The procedure for the GSSG application using the NWK-GSH01 assay is essentially the same as thestandard method, with the following exceptions:
The calibrators are made up using an MPA:Assay Buffer solution (2:1) instead of
Assay Buffer.
C alibrators are treated with 4VP reagent the same as samples
4 N Sodium hydroxide (NaOH) is required but not provided.
Reagent grade methanol or ethanol is required but not provided
4-Vinylpyridine is required but not provided.
Extended reaction time

Reagent Preparation
Prepare 4 N NaOH
Add 15 mL water to flask
Add 5.2 mL 50% NaOH (19.4 N) to 25 ml volumetric flask
Mix
C ool to room temperature
Bring volume to 25 mL with water Store in a plastic bottle at room temperature
orAdd 4 g NaOH pellets to 25 ml volumetric flask
Add 20 mL water volumetric flask
Mix until dissolved
C ool to room temperature
Bring volume to 25 mL with water Store in a plastic bottle at room temperature

Prepare calibrators in a 2 to 1 MPA:Assay Buffer Solution
0 uM GSSG - MPA:Assay Buffer
5 uM GSSG - 25 uL calibrator concentrate in 975 uL MPA:Assay Buffer Solution
10 uM GSSG - 50 uL calibrator concentrate in 950 uL MPA:Assay Buffer Solution
Note: C alibrators will be treated and assayed the same as the samples except they don't
need to be frozen.

Prepare 4-Vinylpyridine

4-vinylpyridine is not supplied with the NWK-GSH01 assay but is available commercially (Aldrich #V3204 or equivalent, C AS 100-43-6). The amount of 4-VP required per assay is small; therefore it isrecommended that one purchase the minimum quantity of reagent.
4-VP is typically stored at -20°C and must be brought to ambient temperature before opening thecontainer.
Add 1 mL of methanol or ethanol to a glass amber vial or other suitable vial with closure.
Add 60 µL 4-VP to produce an approximate 500 mM 4VP solution.
C lose vial and store on ice.
The diluted 4-VP should be used on the same day of prepararation.

Test 4-VP solution (optional)

4-Vinylpyridine darkens with age without affecting the purity (Sigma-Aldrich Specification Sheet). The
activity of 4-VP can be verified by taking advantage of the reaction of DTNB with GSH. Note: The readyto-use DTNB Reagent is supplied with sufficient volume to perform one 4-VP verification.
1. Prepare a 10 mM solution of GSH in Assay Buffer or equivalent buffer at pH 7-8Sample

GSH Buffer 4-VP

Positive C ontrol, µL

1008000Test, µL

100800100900

10Negative C ontrol, µL

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. C alculate the %GSH remaining. (Test-Negative C ontrol)/(Positive C ontrol - Negative C ontrol)
The %GSH remaining should be <1%.

5% MPAnwlss.com/products/pdf/gsh01gssgapp051712.htm

2/3

13/04/03

Oxidized Glutathione (GSSG Application)

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

Sample Processing
Below is a recommended sample processing procedure for tissue samples. Other sample types mayrequire a modified procedure. In the case of whole blood, the sample would be directly extracted in 5%MPA. Note that the assay requires 200 µL clarified homogenate supernatant that has been extracted with
MPA solution (2:1 MPA/Assay Buffer) to measure both GSH and GSSG.
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.
3. Transfer the sample homogenate to a microcentrifuge tube.
4. C entrifuge at 10,000 xg for 2 minutes (or sufficient to produce a clear supernatant)
5. C ollect the supernatant and aliquot an appropriate portion for protein analysis.

Extract Clarified Sample Homogenates in MPA to Precipitate Proteins Prior toAssay
1. Add 150 µL clarified sample homogenate (supernatant) to microcentrifuge tube.
2. Add 150 µL 5% MPA to microcentrifuge tube.
3. Vortex (5 count).
4. C entrifuge at 10,000 xg for 2 minutes (or sufficiently to pellet the precipitate)
5. Place the extract on ice or store at -20°C until assay. (Use undiluted extract for GSSG testing
below or dilute extract 1/20 before testing for "total GSH" according to standard procedure, page
8 of NWK-GSH01 product insert.

pH Adjustment and Treament with 4VP Scavenger Reagent
1. Add 150 µL undiluted Sample Extract or C alibrator to a microcentrifuge tube.
2. Add 7.5 µL 4 N NaOH, mix. (This is necessary to bring the Sample Extract and C alibrators to near
neutral pH before addition of 4-VP scavenger reagent
3. Add 10 µL diluted 4-VP (525 mM), mix. (4VP will be 9.7 mM in final reaction mix
4. Add 350 µL Assay Buffer, mix.
5. Incubate at room temperature for 1 hour.
6. Proceed with the assay protocol below without additional dilution of sample extracts orcalibrators.

Assay
1. Remove microplate from plastic bag.
2. Bring all reagents to room temperature.
3. Add 50 µL of calibrator or sample extract to a designated well.
4. Add 50 µL of DTNB to well.
5. Add 50 µL of GR to well.
6. Incubate microplate for 3-5 minutes at room temperature.
7. Add 50 µL of reconstituted NADPH to wells containing calibrator, control or sample.
8. Begin recording the absorbance at 405 nm (412 if possible) at 15-20 second intervals for 5minutes.
9. Determine the concentration of the controls and samples. If using data reduction on the platereader, skip steps a-c.Important Note:
a. C alculate the rate for each calibrator, control and sample from the linear regression ofA 405 as a function of time.
C alculate the linear regression parameters to obtain the equation of the line.
b. C alculate the concentrations of the controls and samples.
c. C orrect the control and samples for dilution and report results.

^ back to the top ^©2004 Northwest Life Science Specialties, LLC. A ll rights reserved. NWLSSTM , nwlifescience.comTM andthe Northwest Life Science Specialties compass logo are trademarks and/or service marks of Northwest
Life Science Specialties, LLC.
Northwest Life Science Specialties, LLC
16420 S.E. McGillivray, Suite 103, PBM 106, Vancouver, WA 98683
Phone: (360) 449-3091 or (888) 449-3091, Fax: (360) 449-3092, E-mail: nwinfo@nwlifescience.com

nwlss.com/products/pdf/gsh01gssgapp051712.htm

3/3