Using Synthetic Peptide Substrates to Measure Drosophila Caspase Activity

Central to the apoptotic pathway is the activation of caspases that are members of a highly conserved family of cysteine proteases. Caspases are synthesized as inactive zymogens and are generally activated by proteolytic cleavage to form the catalytically active enzyme. Caspase activity in apoptotic cells can be measured by assessing the cleavage of commercially available synthetic caspase substrates. The synthetic substrates contain a caspase cleavage site conjugated to a fluorochrome, such as 7-amino-4-methylcoumarin (AMC), or a chromophore, such as p-nitroaniline (pNA), for colorimetric detection. Here, we present a protocol for the measurement of caspase activity in Drosophila cell extracts by cleavage of the target peptide in the synthetic substrate that releases a fluorochrome or color-producing agent. The signal is measured by a spectrophotometer, with the intensity of the signal being proportional to the amount of substrate cleaved.