[42] Protein S-thiolation and dethiolation

Publisher Summary S-Thiolated proteins—mixed disulfides of proteins and low molecular weight thiols—are very early products of protein oxidation during oxidative stress, occurring within seconds after the generation of oxygen radicals. As a result, assessment of the extent and specificity of this process during oxidative stress is one of the best measures of the primary effects of oxygen radical generation on intact cells. The development of methods for measuring the S-thiolation status of individual proteins, eventually studying organs of intact animals and even humans, is essential for a more complete understanding of the role of oxidative stress in human disease. This chapter introduces a method to quantify the S-thiolation status of mixed cellular proteins in intact cells and second method to determine the activity of the cellular reductive processes—namely, dethiolases. This method tests the effects of toxic materials on protein S-thiolation. Protein dethiolation can be measured with S-thiolated substrate proteins containing a radioactive low molecular weight thiol.