[8] Caspase assays

This chapter describes methods for determining caspase activity using synthetic substrates and natural substrates. It also discusses the method to determine the active caspase concentrations. Although the function of proteases is to hydrolyze peptide bonds in natural proteins, most assays designed to characterize or discover proteases rely on synthetic peptide substrates that are engineered to adapt to specific sites in the enzyme active site. Usually a reporter group is attached to the peptide on the C-terminal side of the scissile peptide bond. Cleavage of the synthetic substrate releases a chromogenic or fluorogenic group that is readily detected by spectroscopic instruments. Thus, these synthetic substrates are conventionally used to define the activities of proteases. Similar strategies are used to synthesize specific protease inhibitors, where the leaving group of the substrate is replaced by a group that reacts with the catalytic machinery of the protease. Activity of the caspases, either purified or in a crude mixture, can be readily measured with synthetic substrates.