Measurement of mixed disulfides including glutathionylated proteins

Mixed disulfides between protein cysteines and low-molecular-weight thiol cysteine or glutathione lead to the formation of cysteinylated proteins or glutathionylated proteins. These types of posttranslational modification are of great importance in the so-called redox regulation, by which changes in the redox state of the cell regulate a number of biochemical processes. We describe the methods for quantitatively measuring the various redox states of cellular thiols including protein cysteines and these mixed disulfides. These include spectrophotometric methods, which do not distinguish between protein-cysteine and protein-glutathione disulfides, and HPLC methods that make such distinction. Finally, we report a method for labeling proteins susceptible to glutathionylation with biotin, to allow their visualization by Western blot after electrophoretic separation, which is used to identify proteins undergoing this posttranslational modification.