Oxidized glutathione assay

1. A method for determining GSH:GSSG ratio in a sample, the method comprising:

(a) providing a first portion of the sample and a second portion of the sample;

(b) contacting the first portion of the sample with a sulfhydryl modifying agent to provide a modified first sample portion;

(c) adding to the modified first sample portion an excess of a reducing agent, glutathione-S-transferase, and a substrate for a glutathione-S-transferase, which is converted to a signal-generating compound in the presence of GSH and glutathione-S-transferase, wherein the reducing agent inactivates the sulfhydryl alkylating agent and reduces any GSSG to GSH, to form a first solution;

(d) measuring the signal of the first solution, wherein neither the inactivated sulfhydryl alkylating agent nor the reducing agent is removed from the first solution;

(e) contacting the second portion of the sample with glutathione-S-transferase and a substrate for a glutathione-S-transferase, which is converted to a signal-generating compound in the presence of GSH and glutathione-S-transferase to form a second solution;

(f) measuring the signal of the second solution; and

(g) comparing the signal of the second solution to the signal of the first solution to determine the GSH:GSSG ratio.

2. The method of claim 1 wherein the sample comprises a cell, media, plasma, serum, blood, or tissue extract.

3. The method of claim 1 further comprising contacting the sample with a lysing agent prior to step a.

4. The method of claim 1 wherein the signal from the first solution and the signal from the second solution is fluorescence.

5. The method of claim 1 wherein the signal from the first solution and the signal from the second solution is luminescence.

6. The method of claim 1 wherein the substrate is a luciferin derivative, which is converted to luciferin in the presence of GSH and glutathione-S-transferase.

7. The method of claim 1 wherein the substrate is a compound of formula (III)

wherein X is N or O;

wherein R 1 , R 2 , R 3 , R 4 and R 5 are independently H, C 1-6 alkyl, CF 3 , halogen, NO 2 , CO 2 R, wherein R is H or C 1-6 alkyl, or any two adjacent R 1 -R 5 can form a fused ring provided that at least one of R 1 , R 3 or R 5 is NO 2 and not all three are NO 2 .

8. The method of claim 7 wherein the fused ring is benzo, naphtho, or heterocyclic.

9. The method of claim 1 wherein the substrate is a compound of formula

10. The method of claim 1 wherein the sulfhydryl modifying agent is N-ethylmaleimide (NEM).

11. The method of claim 1 wherein the reducing agent is dithiothreitol (DTT).