Blot-based detection of dehydroalanine-containing glutathione peroxidase with the use of biotin-conjugated cysteamine.
Dehydroalanine (DHA), alpha,beta-unsaturated amino acid, is found in the position corresponding to the serine, cysteine, and selenocysteine (Sec) residues of various proteins. Proteinaceous Sec is readily oxidized and subsequently undergoes beta-elimination to produce DHA. Glutathione peroxidase (GPx), which contains a Sec at the active site, is irreversibly inactivated by its own substrate as the result of the oxidation of selenium atom followed by the conversion of oxidized Sec to DHA. We developed a convenient method for estimation of the amount of DHA-GPx1 in cell homogenates. This blot-based method depends on specific addition of biotin-conjugated cysteamine to the DHA residue followed by detection of biotinylated protein based on its interaction with streptavidin. The method required an immunoprecipitation of GPx1 before labeling with the cysteamine derivative because many other proteins contain DHA. With the use of this method, we found that conversion of the Sec residue at the active site of GPx1 to DHA occurred during aging of red blood cells (RBCs) in vivo as well as in RBCs exposed to H(2)O(2) generated either externally by glucose oxidase or internally as a result of aniline-induced Hb autoxidation. Accordingly, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime of 120 days. Previous studies suggested that the activity of GPx1 in RBCs is most influenced by lifestyle and environmental factors such as the use of dietary supplements and smoking habit. Therefore, DHA-GPx1 in RBCs might be a suitable surrogate marker for evaluation of oxidative stress in the body. Our blot-based method for the detection of DHA-GPx1 will be very useful for evaluation of such stress. In addition, similar blot detection method can be devised for other proteins for which immunoprecipitating antibodies are available.
Rhee
Sue Goo
SG
Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu, Seoul, Korea.
Cho
Chun-Seok
CS
D000409:Alanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001710:Biotin / Q000737:chemistry*; D002478:Cells, Cultured; D003543:Cysteamine / Q000737:chemistry*; D005979:Glutathione Peroxidase / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D047468:Immunoprecipitation / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D018796:Immunoconjugates; D003543:Cysteamine; D001710:Biotin; D019809:Streptavidin; C015102:dehydroalanine; D005979:Glutathione Peroxidase; D000409:Alanine