Enzymatic assays

The serine protease activity of the whole protein extract, 5 µg of total protein, was assessed in activation buffer (Tris–HCl [10 mM], pH 7.5) using a specific fluorogenic peptide substrate, Z-FR-AMC [1 mM], at a final volume of 60 μL. Samples were incubated (37 °C, 60 min), and the variance in the relative fluorescence was monitored on a Molecular Devices SpectraMax spectrophotometer (Gemini XPS).

Inhibition assays were performed by incubation (25 °C, 5 min) of each sample with specific inhibitors of proteases: E-64 [10 mM] (for cysteine proteases), PMSF [1 mM] (for serine- and cysteine proteases), AEBSF [1 mM] (for serine proteases such as trypsin, chymotrypsin, plasmin, kallikrein and thrombin), and TLCK [100 mM] (for serine proteases such as trypsin and trypsin-like). All inhibitors were assessed at the maximum recommended concentrations⁶¹.

The substrate cleavage rate was defined as follows: v = Δs/Δt, where v = velocity, Δs = substrate concentration variation and Δt = total reaction time, as determined elsewhere³⁹. The self-degradation of the fluorescent peptide substrate was controlled throughout the assay to avoid incorrect readings; the enzymatic activity is expressed as mmol min⁻¹.mg of protein⁻¹.