Measurement of Protein Glutathionylation

Proteins contain free, exposed thiols that can be glutathionylated in the native state as a result of thiol-disulfide exchange reactions with glutathione disulfide, catalyzed by glutaredoxin. A number of other reactions can also lead to protein glutathionylation. The modification of proteins by glutathionylation is important in oxidative damage and may be an important post-translational modification to proteins involved in redox signaling. This unit describes methods for the identification of glutathionylated proteins and quantification of the extent of glutathionylation. The protocols described use isolated mitochondrial protein complexes, mitochondrial membranes, and intact mitochondria, but can be easily adapted to other systems.