Protease assays using synthetic fluorogenic peptides

Short fluorogenic peptides are a fast and simple method to measure protease enzymatic activity and are commonly used to study substrate specificity using peptides with different sequences. Here, the protease activity was monitored by the hydrolysis of the fluorophore 7-amino-4-methyl coumarin (AMC) from the synthetic peptide substrates Z-Val-Leu-Lys-AMC and Tos-Gly-Pro-Arg-AMC (Z and Tos correspond to Carboxybenzyl and Tosyl, respectively; the blocking groups that enable cathepsin endopeptidases to position for peptide hydrolisis). The kinetic parameters were determined in a reaction buffer containing 0.1 M sodium phosphate buffer, pH 6.0, 1 mM DTT and 1 mM EDTA at 25°C with final enzyme concentrations in the 10⁻⁹ M range. Different substrate concentrations (5–100 μM) were added after a 10 min pre-incubation of the enzyme in reaction buffer and reaction rates were measured in duplicate. The slope of the progress curves were obtained by continuous recording in a FluoStar spectrofluorimeter at 345 nm excitation and 440 nm emission wavelengths, using an AMC standard curve for product concentration calculation. The enzyme concentration was determined by active-site titration with E-64c. The kinetic parameters V_max and K_M were estimated by non-linear regression analysis of the Michaelis–Menten plot using the OriginPro 6.1 software. k_cat was calculated as V_max/[E] where [E] is the active enzyme concentration (fit to the Michelis-Menten equation for FhCL1 hydrolysis of Z-Val-Leu-Lys-AMC peptide is included as an example in Supplementary Table 2). The FhCL2 recombinant enzyme for the kinetic analysis was kindly provided by Prof. John Dalton (School of Biological Sciences, Queen's University Belfast).