Measurement of Oxidized/Reduced Glutathione Ratio - Protein Misfolding and Cellular Stress in Disease and Aging - page 260

5. GSH Enzyme Mixture (Caymen Chemical, Ann Arbor, MI)
contains glutathione reductase and glucose-6-phosphate
dehydrogenase in 200 mL of buffer. Add 2 mL of MES buffer
(from step 2) to vial and vortex. Solution is stable at 0-4°C
for 2 weeks.

2.3. Experiment 2
GSSG Determination

1. 2-Vinylpyridine (1 M) solution dissolved in ethanol.
2. All reagents from Subheading 2.2 (see Note 1).

3. Methods

Sample preparation for the GSH/GSSG ratio assay is critical and
is dependent upon tissue and/or bodily fluid type ( 15 ). Tissues
low in g-glutamyl transpeptidase concentration, such as brain, can
be frozen at −80°C and assayed for GSH/GSSG at a later date.
However, tissues high in g-glutamyl transpeptidase, such as the
kidneys and pancreas, should be processed immediately upon sac-
rifice and never frozen. The thawing process accelerates g-glutamyl
transpeptidase activity, potentially leading to misleading results.
Bodily fluids, such as plasma, must also be processed immediately
after isolation because GSH oxidation occurs rapidly compared to
tissue. However, once plasma has been processed, the aliquot can
be frozen at −80°C (See Note 2).
Measuring the GSH/GSSG ratio can be accomplished using
a UV-Visible spectrophotometer equipped with a 96-well plate
reader. Such instrumentation is the best way to measure the
GSH/GSSG ratio with multiple samples. Two separate experi-
ments, that is, measuring total GSH levels followed by measuring
GSSG levels, are required to determine the GSH/GSSG ratio.
Experiment 1: Measuring total GSH. Measuring total GSH
utilizes the reaction of GSH and GSSG with Ellman's reagent
(DTNB) to produce 2-nitrobenzoic acid anion (TNB − ), a com-
pound containing a chromophore that absorbs at 412 nm ( 16 ).
These reactions are catalyzed by glutathione reductase, which
requires NADPH as a cofactor. (To supply the reactions with
NADPH, glucose-6-phosphate (G6P) and glucose-6-phosphate
dehydrogenase (G6PDH) are present in the enzyme mixture
mentioned in Subheading 2.2 .) Since both GSH and GSSG
react with DTNB to form TNB − , an intensely colored reagent
that absorbs at 412 nm, the total level of GSH is measured in
the cell.
Experiment 2: Measuring GSSG. To measure GSSG only,
reduced GSH is derivatized with 2-vinylpyridine ( 17 ). This modi-
fication ensures that GSSG is the only form of glutathione that
can react with the DTNB reagent. The GSSG level is subtracted