Peptide Hydrolysis

Peptidase activity was monitored by the rate of production of fluorescent AMC (7-amino-4-methylcoumarin) after cleavage from fluorogenic peptide substrates. The peptide substrates used in the hydrolysis assay were N-succinyl-Leu-Tyr-AMC (Suc-LY-AMC) as substrate 1 (S1) and Suc-LLVY-AMC as substrate 2 (S2) (Sigma). Peptidase assays were performed in black flat-bottom 96-well plates (Invitrogen) at 37 °C. Each 96-well contained a fluorogenic peptide substrate (0.1 mM) and pure rClpP isoforms or its mixture (0.0125 μg μL^–1) in 80 μL of buffer-F (50 mM phosphate buffer, 100 mM KCl, 5% glycerol; pH 7.6). Fluorescence was measured in the Infinite M200Pro plate reader (Tecan) at 380 and 460 nm wavelength of excitation and emission, respectively. The peptide substrate S1 (0.01–5.0 mM) was incubated with rClpP isoform mixture (0.025 μg μL^–1) in 80 μL of buffer-F to determine the kinetic parameters of leptospiral rClpP isoform mixture. The subsequent experimental procedure to detect the hydrolysis of substrate S1 was the same as described above. Initial and final readings were taken at 0 and 1 h, respectively (excitation: 380 nm/emission: 460 nm). The measurements obtained were processed in Microsoft Excel and then data were transferred to Origin9.0 for Hill kinetics and statistical analysis. Similar peptidase assays were carried out on substrates S1 and S2 using pure rClpP mutant isoforms (rClpP1S98A and rClpP2S97A) or the rClpP isoform mixture constituted by either of the mutant isoforms. All the experiments were performed twice independently and in duplicates.