Quantitation of cytochrome c release from rat liver mitochondria

The apoptogenic protein cytochrome c can be quantitated by reverse-phase HPLC, but this method is not utilized by those who investigate mechanisms of cell death. Here, we extend the sensitivity of the method to exceed that available from immunogenic approaches and report specific procedures for applying the method to preparations of intact mitochondria, and to supernatants and pellets that arise from mitochondrial incubations. The detection limit corresponds to 0.6% of total cytochrome c found in 100 microg of rat liver mitochondrial protein, or to all of the cytochrome c that is expected in approximately 6000 hepatocytes. A single determination can be completed in 20 min, compared to a time scale of days for Western blotting methods, or hours for ELISA-based methods. The procedures are illustrated by experiments that determine the amount of cytochrome c released following the mitochondrial permeability transition as a function of medium ionic strength, and by long-term incubations of intact mitochondria in the presence and absence of an exogenous oxidizable substrate. Swelling and the release of adenylate kinase activity have been determined simultaneously to show how the data can be applied to evaluate the role of outer membrane disruption in mechanisms that release cytochrome c.