Measuring the redox state of cellular peroxiredoxins by immunoblotting

The peroxiredoxins (Prxs) are a family of thiol peroxidases that scavenge hydroperoxides and peroxynitrite. The abundance and reactivity of these proteins makes them primary targets for cellular H(2)O(2). The catalytic cycle of typical 2-Cys Prxs involves formation of an intermolecular disulfide bond between peroxidatic and resolving cysteines on opposing subunits. Rapid alterations in the ratio of reduced monomer and oxidized dimer have been detected in the cytoplasm and mitochondria of cultured cells exposed to various exogenous and endogenous sources of oxidative stress. Here we describe immunoblot methods to monitor the interconversion of individual 2-Cys Prxs in cultured cells. We also outline an adaptation of this method to measure the extent to which individual 2-Cys Prxs become hyper oxidized in treated cells. Together, these methods enable the redox status of cellular Prxs to be assessed and quantified in a rapid and robust manner.