4.4. Dipeptidyl Peptidase IV Inhibitory Assay and Kinetics Assays

The DPP-IV inhibitory activities of the substance after ultrafiltration and the peptides identified (synthesized by Chinese Peptide Company, Hangzhou, China; purity of 96–97%) were evaluated. The DPP-IV inhibition assay was performed as described by Velarde-Salcedo et al. [50], with slight modifications. The assay was performed in 96-well microplates, and 100 μL of enzyme (final concentration 1.9 U/L) was preincubated with the samples (the concentrated ultra-filtrate mentioned above and the synthesized peptide, with concentrations from 62.5–1500 μg/mL) at 37 °C for 15 min prior to the initiation of the reaction, by adding 50 μL of Gly-Pro p-nitroanilide (final concentration 0.29 mM). The final volume of the reaction system was 200 μL, and the reaction was conducted in 100 mM Tris-HCl buffer (pH 7.6). The absorbance was obtained at 405 nm in a microplate reader (Synergy H1, BioTek Instruments Inc., Winooski, VT, USA), after incubation at 37 °C for 1 h.

The most potent peptide (peptide II) was synthesized and further studied for its modes of action on DPP-IV using kinetics assays. The initial rate of hydrolysis of the substrate Gly-Pro p-nitroanilide by DPP-IV was measured using substrate concentrations of 0.29, 0.58, 1.16, 2.32, and 4.64 mM (final assay concentrations) in the absence (100 mM Tris-HCl buffer, pH 7.6) and presence of different concentrations of the peptide II (1.02 and 2.04 mM, respectively). The substrate (50 μL), with or without an inhibitor (50 μL), was pre-incubated at 37 °C for 15 min, and the reaction was started by the addition of 100 μL of the DPP-IV enzyme (final concentration: 1.9 U/L). The absorbance of the released p-nitroanilide was monitored at 37 °C and 405 nm for 60 min using a microplate reader (Synergy H1, BioTek Instruments Inc.). Lineweaver–Burk plots of DPP-IV activity in the absence and presence of peptide II were created to show the modes of inhibition.