3.10. Stability of Synthesized DPP-IV Inhibitory Peptides

The synthetic peptide was pre-incubated with DPP-IV to determine stability against DPP-IV. Based on this peptide stability, inhibitors of DPP-IV were classified into substrate, prodrug, or true inhibitor type. According to the previous method by Nongonierma et al. [37] with slight modification, 50 μL of peptides (500 µM) dissolved in 1% DMSO were pre-incubated with 100 μL of DPP-IV (1 U/μL) in DPP-IV buffer at 37 °C for 3 h. Consequently, 75 µL of pre-incubation mixture was combined with 25 µL of 2 mM NaOH and 25 µL of 1.6 mM Gly-Pro-p-nitroanilide, and further pre-incubated at 37 °C for 10 min. The mixture was added with 25 µL of DPP-IV buffer and then incubated at 37 °C for 60 min. The reaction was ended using 100 µL of 1M sodium acetate at pH 4.0. The absorbance of each reaction was measured at 405 nm in a SpectraMax 190 Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA) to determine DPP-IV inhibitory activity of peptide after a pre-incubation period. The remaining 75 µL of the original mixture was heat-inactivated at 95 °C for 10 min to end enzyme activity after which it was injected into LC-MS to confirm the stability of the peptide.