Enzymatic activity determinations

Enzymatic activity of the recombinant rLbDPP3 or rTF-LbDPP3 was evaluated with the fluorogenic DPP3 assay kit (BPS Bioscience, Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, the reaction (100 μl) was performed in black 96-well microplates using 200 ng (1.5 μl) of the rLbDPP3 and 50 ng of the recombinant N-GST-Tag human enzyme (rhDPP3) (1 μl), provided in the kit, as positive control. The Z-Arg-Arg-AMC was used as substrate in a buffer suitable for the enzyme, provided by the kit. The production of 7-amino-4-methylcoumarin (AMC) was measured every 2 min in a fluorometer (Fluostar Optima) at a wavelength of 355 nm for excitation and 460 nm for emission during 1 hour and 30 min. The substrate affinity of rhDPP3 and rLbDPP3 was measured by testing the enzymatic activity in the presence of different Arg-Arg-AMC concentrations, ranging from 5 to 50 μM for rhDPP3 and 20 to 160 μM for rLbDPP3. The concentration of free AMC produced in the enzymatic reaction was assessed using a standard curve with known concentrations of AMC. The Michaelis-Menten constant (Km) and the maximum rate of the reaction (Vmax) were assessed using the models of Monod, plotting the substrate concentration (μM) and velocity of the reaction in nM/min, and the Langmuir equation: substrate concentration (μM) and the ratio between substrate concentration and velocity (S/V, μM(nM/min)^-1). Briefly, the Vmax was defined as the inverse of the slope of the Langmuir linear regression equation and the Km was calculated multiplying the Vmax by the intersect of the same equation. The turnover number (kcat) was calculated as Vmax/enzyme concentration. The effects of the tynorphin and IVYPW inhibitors (synthesized by the Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Chile) on the activity of both human and L. braziliensis enzymes were also evaluated. For this purpose, the enzymatic activity was performed as described previously, but in the presence or absence of a 3-fold molar excess of each inhibitor dissolved in water. The enzymatic activity assays were repeated twice for the evaluation of kinetic parameters (Km and Vmax), and at least thrice for the inhibition experiments, each measure was done in triplicate.