Enzymes included bovine trypsin, bovine chymotrypsin, human cathepsin B, human cathepsin L, and human cathepsin S (all Sigma-Aldrich) and human cathepsin K (Enzo Life Sciences). Purified F. hepatica cathepsin L1 (FhCL1), F. hepatica cathepsin L2 (FhCL2) and F. hepatica cathepsin L3 (FhCL3)66 (link) and recombinant FhKT1 and FhKT1Leu15/Arg1510 (link) were expressed as active recombinant proteins in P. pastoris. Reaction conditions and substrates employed for measuring the activity of each protease were as reported by Smith et al.10 (link). Additionally, rFhCL3 activity was measured using the fluorogenic substrate Z-Gly-Pro-Arg-NHMec (20 μM).
KT inhibitors (2 μM) was incubated with each protease in a 100 μl volume of reaction buffer for 15 min at 37 °C. Reaction were brought to 200 μl with the addition of fluorogenic substrate dissolved in reaction buffer and proteolytic activity measured as RFU (relative fluorescent units) using a PolarStar Omega spectrophotometer (BMG LabTech, UK). Inhibition constants were determined using the Morrison equation for tight-binding inhibition as previously described10 (link).
KT inhibitors (2 μM) was incubated with each protease in a 100 μl volume of reaction buffer for 15 min at 37 °C. Reaction were brought to 200 μl with the addition of fluorogenic substrate dissolved in reaction buffer and proteolytic activity measured as RFU (relative fluorescent units) using a PolarStar Omega spectrophotometer (BMG LabTech, UK). Inhibition constants were determined using the Morrison equation for tight-binding inhibition as previously described10 (link).
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