For the calculation of active site concentrations, Ac-DEVD-AFC was used for caspases-3 and -7 according to the same protocol, and different amounts of zVAD-fmk were added to all reactions. The ratio between reaction velocity with inhibitor (Vi) over reaction velocity without inhibitor (V0) was plotted against the inhibitor concentration, and the concentration for y = 0 was determined as the active site concentration of the caspase.