Dethiolation of protein mixed-disulfides

The dithiol proteins, glutaredoxin, thioredoxin, and protein disulfide isomerase, were examined as dethiolases (i.e., reductases for protein mixed-disulfides) by studying the specificity and reactivity for an S-glutathiolated protein mixture. The 35S-glutathiolated protein mixture was prepared from 35S-labeled rat hepatocytes by diamide treatment. Dethiolation of individual 35S-labeled proteins was analyzed by combining SDS-PAGE and autoradiography. The dithiol proteins greatly enhanced dethiolation rates and could completely dethiolate all of the S-glutathiolated proteins. The dethiolation rate for individual proteins by each dithiol protein was compared and glutaredoxin was the most effective for every S-glutathiolated hepatocyte protein. When testing the reduction of insulin disulfide, we found that glutaredoxin did not catalyze insulin reduction, while protein disulfide isomerase and thioredoxin did. This suggests that glutaredoxin may be specific for S-glutathiolated proteins. The redox potential of glutaredoxin was determined to be -0.159 ± 0.004 V, indicating that glutaredoxin was thermodynamically a weaker reductant than E. coli thioredoxin and similar to protein disulfide isomerase. The effective dethiolation by glutaredoxin compared to thioredoxin and protein disulfide isomerase, therefore, might be determined by kinetic factors. Glutathione-binding at the active site of glutaredoxin was suggested as an important factor for the effective dethiolation. The results suggested that glutaredoxin might be the major dethiolase for S-glutathiolated proteins in cells;Dethiolation of the mixture of 35S-glutathiolated hepatocyte proteins by glutathione was also studied. All the 35S-glutathiolated proteins were dethiolated by glutathione. Dethiolation of either the mixed hepatocyte proteins or pure S-glutathiolated phosphorylase b was saturable with respect to the concentration of glutathione. The half maximal rate of dethiolation occurred at 0.16 mM GSH. The dethiolation rate decreased in the presence of denaturing agents such as guanidinium chloride and sodium dodecyl sulfate, as well as the glutathione analogs, S-methylglutathione and glutathione sulfate. The saturation kinetics, inhibition by glutathione analogs and protein denaturants, strongly suggest that glutathione forms noncovalent complexes with S-thiolated proteins during dethiolation. This glutathione-binding suggests that liver proteins may contain glutathione-binding sites closely associated with the reactive sulfhydryls that undergo S-thiolation and dethiolation