Glutathione is a sulfur-containing tripeptide, which functions in cellular antioxidant defense mechanisms. It is also thought to have a regulatory effect on enzymes during oxidative stress, through the formation of mixed disulfides in the proteins. Presently, there are no sensitive assays which can detect proteins that have undergone S-glutathiolation. The is partly due to the inability to obtain sensitive antibodies to glutathione because of its ubiquitous nature. As a result, our research focuses on the development of a Western analysis method involving chemical modification of these glutathiolated proteins. The model protein being used is bovine serum albumin. Mercaptoethanol-treated albumin is modified by S-glutathiolation of cysteine residues. The extent of glutathiolation is then measured with Ellman's reagent [2,2,-dithiobis(2-nitrobenzoic acid)] (DTNB). The unreacted sulfhydryl groups are then carboxymethylated, and the protein treated with thiol transferase to release glutathione. This previously S-glutathiolated cysteine is then tagged with 5-iodoacetamidofluorescein (5-IAF). A sodium-dodecyl sulfate polyacrylamide gel electrophoresis/Western analysis allows identification of the tagged proteins. This method should allow us to further investigate protein modification and regulation by glutathione during oxidative stress.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000315-01
Application #
6162655
Study Section
Special Emphasis Panel (LB)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code