A new HPLC method to determine glutathione-protein mixed disulfide

A sensitive method for measuring glutathione-protein mixed disulfides is described. The method is based on cleavage of the protein disulfides with performic acid followed by reaction with dinitrofluorobenzene and HPLC analysis with a Bondapak-amine column. Samples containing 0.1 nmoles or more of glutathione-protein mixed disulfide can be detected. The method has been used to demonstrate (a) the presence of low levels of glutathione mixed disulfide in gamma crystallin isolated from bovine lenses, (b) a dramatic increase in such mixed disulfides after exposure of denatured gamma crystallin to O2 in the presence of glutathione, and (c) the formation of glutathione-protein mixed disulfide in lens epithelial cells exposed to 0.6 mM H2O2 for one hour.