TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on Caspase-3. We designed a TAMRA (5'-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the Caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of Caspase-3, with V(max) of 41.4±3.3 nM/min and K(M) of 1.60±0.23 μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for Caspase-3 inhibitor screening assays or for real-time detection of Apoptosis in living cells.