S-thiolation and irreversible oxidation of sulfhydryls on carbonic anhydrase III during oxidative stress: a method for studying protein modification in intact cells and tissues.

S-thiolation of carbonic anhydrase III (CA III) in cultured rat hepatocytes under oxidative stress was studied by immunodetection on nitrocellulose blots of isoelectrofocusing gels. In cells treated with menadione, three S-thiolated forms of CA III were detected, whereas only two forms were observed in hepatocytes treated with t-butyl hydroperoxide. Two "nonreducible" oxidized forms of CA III were also detected on nitrocellulose blots. These forms increased with the amount of stress and were the only modified forms of CA III in buthionine sulfoxide-treated hepatocytes containing 10-fold less glutathione than control hepatocytes. These experiments support the concept that S-thiolation protects CA III from irreversible oxidation during oxidative stress. Partly and fully S-thiolated forms of CA III were easily detected in both male and female hepatocytes by the immunoblotting method, although female cells contained 15-fold less CA III than did male liver. S-thiolated forms of CA III were also detected in rat skeletal muscle and heart showing the utility of this method for determining the effect of oxidative stress on specific S-thiolatable protein in several tissues in vivo.