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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
- Xu, Feiran, Yao, Yijun, Xu, Xiaoying, Wang, Mei, Pan, Mengmeng, Ji, Shengyang, Wu, Jin, Jiang, Donglei, Ju, Xingrong, Wang, Lifeng
- Journal of agricultural and food chemistry 2019 v.67 no.13 pp. 3679-3690
- Brassica napus, amino acids, chemical bonding, computer simulation, enzyme inhibition, gel chromatography, hydrolysates, inhibitory concentration 50, mutation, peptides, protein domains, rapeseed, reversed-phase high performance liquid chromatography, subtilisin, tandem mass spectrometry, trypsin, ultrafiltration
- 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.