Cytochrome c binding to Apaf-1: the effects of dATP and ionic strength

In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. Here, we show through fluorescence polarization experiments that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c.Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c.Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. We also report here that the cytochrome c.Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins.