Identification and Quantification of DPP-IV-Inhibitory Peptides from Hydrolyzed-Rapeseed-Protein-Derived Napin with Analysis of the Interactions between Key Residues and Protein Domains

Previously reported peptides derived from napin of rapeseed (Brassica napus) have been shown to inhibit DPP-IV in silico. In the present study, napin extracted from rapeseed was hydrolyzed by commercial enzymes and filtered by an ultrafiltration membrane. The napin hydrolysate was then purified by a Sephadex G-15 gel-filtration column and preparative RP-HPLC. A two-enzyme-combination approach with alcalase and trypsin was the most favorable in terms of the DPP-IV-inhibitory activity (IC50 = 0.68 mg/mL) of the napin hydrolysate. Three peptides and one modified peptide (pyroglutamate mutation at the N-terminus) were identified using HPLC-triple-TOF-MS/MS. DPP-IV-inhibitory activity and the types of enzyme inhibition were also determined. Meanwhile, key residues associated with the interactions between the selected peptides and DPP-IV were investigated by molecular docking. IPQVS has key amino acid residues (Tyr547, Glu205, and Glu206) that are consistent with Diprotin A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of DPP-IV.