3.3. Assay of Human DPP III Activity

Purified hDPP III (1.5 nM) was preincubated with coumarin derivatives (10 µM) first for 5 min at 25 °C and then for 10 min at 37 °C in 50 mM Tris-HCl buffer, pH 7.4. The enzymatic reaction was started with Arg₂-2NA (40 μM) as a substrate, and after the 15 min incubation at 37 °C the reaction was stopped and the absorbance was measured using the spectrophotometric method described before [41]. Percentage enzyme inhibition (% inh.) was calculated by comparing the enzymatic activity without (control activity), and with inhibitor (inhibited activity) using the following formula:

The IC₅₀ values of selected compounds (12 and 36) were determined by the linear regression of the percentage of enzyme inhibition against the increasing concentrations (0.5–3.5 μM) of coumarin derivatives. The IC₅₀ value is defined as the concentration of an inhibitor that caused a 50% reduction in the enzyme activity under assay conditions. The stock solutions (8 mM) of coumarin derivatives were freshly prepared in dimethyl sulfoxide and diluted with 50 mM Tris-HCl buffer, pH 7.4 buffer before assay of enzymatic activity.