Apoptosis can be mediated by mechanisms other than the traditional caspase-mediated cleavage cascade. There is growing recognition that alternative proteolytic enzymes such as the lysosomal cathepsin proteases may initiate or propagate proapoptotic signals. Cathepsins are lysosomal enzymes that are also used as sensitive markers in various toxicological investigations. The Cathepsin-L Activity Assay kit is a fluorescence-based assay that utilizes the preferred cathepsin-L substrate sequence FR labeled with AFC (amino-4-trifluoromethyl coumarin). Cell lysates or other samples that contain cathepsin-L will cleave the synthetic substrate FR-AFC to release free AFC.

Figure: a. Measurement of recombinant human Cathepsin L activity. b. Measurement of Cathepsin L activity in HepG2 lysate. c. Measurement of Cathepsin L activity in untreated and treated (5 or 25 µM Acetaldehyde) HepG2 lysates. Lysate without the addition of substrate was used as background control. Free AFC was used to obtain AFC standard curve. The protein amount in lysate obtained after treatment with 25 µM of acetaldehyde was lower as compared to untreated or 5 µM treated cells. Assays were performed according to the kit protocol.