GSSG was assayed by measuring 5-thio-2-nitrobenzoic acid
(TNB) which was produced from the reaction of reduced GSH with DTNB.
The sulfhydryl group of GSH reacts with the Elman's reagent [DTNB, (5,5-dithio-bis-2-(nitrobenzoic acid)] and produces a yellow-colored 5-thio-2-nitrobenzoic acid
The recycling assay for total glutathione is oxidized by 5,5-Dithiobis (2 acid) (DTNB) to give GSSG with stoichiometric formation of 5-thio-2-nitrobenzoic acid
. GSSG is reduced to GSH by the action of the highly specific glutathione reductase (GR) and nicotinamide adenine dinucleotide phosphate(reducedform; NADPH).
The yellow product, 5-thio-2-nitrobenzoic acid
(TNB) is measured spectrophotometrically at 412 nm.
For total GSH, GSH was oxidized by DTNB to give GSSG with stoichiometric formation of 5-thio-2-nitrobenzoic acid
Smaller amounts of HOCl can be determined by using dyes such as 5-thio-2-nitrobenzoic acid
(TNB) and 3,3',5,5'-tetramethylbenzidine (TMB).
The reactivity towards hypochlorous acid was determined by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid
oxidation to 5,5'-dithiobis(2-nitrobenzoic acid).
Glutathione concentration was determined by measuring absorbance of TNB (5-thio-2-nitrobenzoic acid
) at 405-414 nm.
The amount of total GSH was determined by formation of 5-thio-2-nitrobenzoic acid
converted from DTNB.