Boc 5 p r amc fluorogenic peptide substrate

The activity of murine kallikrein-related peptidase (KLK)5 (R&D Systems, Minneapolis, MN, USA) and mesotrypsin (PRSS3) (R&D Systems) dissolved in various pH (3.5–8.0) buffers was measured according to the manufacturer’s protocol. Briefly, a murine KLK5 activity assay was performed at 37 °C using 50 mM sodium phosphate buffer (pH 5.8–8.0) or 50 mM acetate buffer (pH 3.5–5.6) containing 0.50 μg/mL mouse KLK5 and 400 μM Boc-V-P-R-AMC Fluorogenic Peptide Substrate (R&D Systems) at the final concentrations. After 30 min of incubation, the fluorescence was measured at an excitation/emission wavelength of 380/460 nm using the PerkinElmer 2030 Multilabel Reader. The mouse PRSS3 activity assay was performed at 37 °C in 50 mM sodium phosphate buffer (pH 5.8–8.0) or 50 mM acetate buffer (pH 4.1–5.6) containing 0.02 μg/mL mouse PRSS3 and 50 μg/mL BODIPY FL casein (Thermo Fisher Scientific). After a 30-min incubation, the fluorescence was measured at an excitation/emission wavelength of 485/535 nm using the PerkinElmer 2030 Multilabel Reader. To determine the relative enzyme activity of murine KLK5 and PRSS3, relative enzyme activity was calculated based on the formula: (A − B)/(C − B) × 100, where A = fluorescence at the indicated pH, B = fluorescence without enzymes, and C = maximum fluorescence obtained in the assay (i.e. pH 7.8 for murine KLK5 and pH 7.8 for PRSS3).