Oxidized glutathione assay

Patent metadata

Field	Value
Publication number	US-9816127-B2
Application number	US-201113222697-A
Country	US
Kind code	B2
Filing date	Aug 31, 2011
Priority date	Sep 1, 2010
Publication date	Nov 14, 2017
Grant date	Nov 14, 2017

Abstract

The present invention provides an assay for detection of oxidized glutathione (GSSG).

First claim

The invention claimed is:

1. A method for detecting GSSG comprising:
a. contacting a sample with a sulfhydryl alkylating agent;
b. contacting the sample with an excess of a reducing agent, glutathione-S-transferase, and a substrate for 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 in the sample to GSH; and
c. detecting the signal generated from step (b), thereby confirming the presence of GSSG in the sample, wherein neither the inactivated sulfhydryl alkylating agent nor the reducing agent is removed from the sample.

2. The method of claim 1 wherein the signal is quantified.

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

4. The method of claim 1 further comprising contacting the sample with a lysing agent in step a.

5. The method of claim 1 wherein the signal is luminescence.

6. The method of claim 1 wherein the substrate for glutathione-S-transferase 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 NO 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 hetrocyclic.

9. The method of claim 1 wherein the sulfhydryl modifying agent is N-ethylmaleimide.

10. The method of claim 1 wherein the reducing agent is DTT.

11. A method of determining oxidative stress in a sample comprising:
(a) contacting the sample with a sulfhydryl alkylating agent;
(b) contacting the sample with an excess of a reducing agent, glutathione-S-transferase, and a substrate for 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 in the sample to GSH; and
(c) measuring the signal, thereby confirming oxidative stress in the sample, wherein neither the inactivated sulfhydryl alkylating agent nor the reducing agent is removed from the sample.

12. The method of claim 11 wherein the sample comprises a cell, media, plasma, serum, blood, or tissue extract.

13. The method of claim 11 further comprising contacting the sample with a lysing agent in step a.

14. The method of claim 11 wherein the signal is luminescence.

15. The method of claim 11 wherein the substrate for glutathione-S-transferase is a luciferin derivative which is converted to luciferin in the presence of GSH and glutathione-S-transferase.

16. The method of claim 11 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 .

17. The method of claim 16 wherein the fused ring is benzo, naphtho, or hetrocyclic.

18. The method of claim 11 wherein the sulfhydryl modifying agent is N-ethylmaleimide.

19. The method of claim 11 wherein the reducing agent is DTT.

20. A method for detecting GSSG comprising:
a. contacting a sample with a sulfhydryl alkylating agent;
b. contacting the sample with an excess of a reducing agent, glutathione-S-transferase, and a substrate for 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 in the sample to GSH; and
c. detecting the signal generated from step (b), thereby confirming the presence of GSSG in the sample, wherein neither the inactivated sulfhydryl alkylating agent nor the reducing agent is removed from the sample, and wherein the signal is fluorescence.

21. The method of claim 20 wherein the signal is quantified.

22. The method of claim 20 wherein the sample comprises a cell, media, plasma, serum, blood, or tissue extract.

23. The method of claim 20 further comprising contacting the sample with a lysing agent in step a.

24. The method of claim 20 wherein the sulfhydryl modifying agent is N-ethylmaleimide.

25. The method of claim 20 wherein the reducing agent is DTT.

26. A method of determining oxidative stress in a sample comprising:
(a) contacting the sample with a sulfhydryl alkylating agent;
(b) contacting the sample with an excess of a reducing agent, glutathione-S-transferase, and a substrate for 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 in the sample to GSH; and
(c) measuring the signal, thereby confirming oxidative stress in the sample, wherein neither the inactivated sulfhydryl alkylating agent nor the reducing agent is removed from the sample, and wherein the signal is fluorescence.

27. The method of claim 26 wherein the signal is quantified.

28. The method of claim 26 wherein the sample comprises a cell, media, plasma, serum, blood, or tissue extract.

29. The method of claim 26 further comprising contacting the sample with a lysing agent in step a.

30. The method of claim 26 wherein the sulfhydryl modifying agent is N-ethylmaleimide.

31. The method of claim 26 wherein the reducing agent is DTT.

Assignees

• Huang Fen
• Klaubert Dieter
• Shultz John
• Zhou Wenhui
• Promega Corp

Inventors

• Huang Fen
• Klaubert Dieter
• Shultz John
• Zhou Wenhui

Classifications

• G01N21/253

  for batch operation, i.e. multisample apparatus (analytical automats G01N35/00) · CPC title

• G01N21/763

  Bioluminescence · CPC title

• G01N2333/91177

  Glutathione transferases (2.5.1.18) · CPC title

• C12Q1/48 Primary

  involving transferase · CPC title