Since the discovery of the key role of cytochrome C in the activation of caspase 9, intense interest has focused on the role of mitochondria in apoptosis/programmed cell death. Mitochondria undergo two major alterations during apoptosis. The first is the permeabilization of the outer mitochondrial membrane. This event is tightly regulated by members of the Bcl-2 family and involves the conformational change of pro-apoptotic family members such as Bax. Second, the electrochemical gradient that is normally present across the inner mitochondrial membrane is lost (membrane depolarization). This event is sometimes mediated by the permeability transition pore (PTP). The order in which these events occur and whether one causes the other has been hotly debated in the literature. Nonetheless, the majority of reports suggest that mitochondria outer membrane permeabilization (MOMP) precedes membrane depolarization. In this chapter, methods that examine membrane depolarization and the conformational change in Bax are described.
