Purification and Characterization of Dipeptidyl Peptidase-Ⅳ from Bovine Kidney

The purpose of this study is to efficiently prepare high-purity dipeptidyl peptidase-Ⅳ (DPP-Ⅳ) from beef kidney by sequential acid precipitation, ammonium sulfate fractionation and gel filtration chromatography. Under reducing conditions, the molecular mass of the purified protein was estimated to be 106 kDa with a purification fold of 169.9 and a yield of 9.5%. Twelve peptides with a total of 517 amino acid residues were identified from the DPP-Ⅳ by mass spectrometry. The matching degree of the 12 peptides with the amino acid sequence of bovine DPP-Ⅳ standard was 100%. The DPP-Ⅳ was found to be a dimeric glycoprotein (210 kDa), with an isoelectric point of 5.8. The secondary structure of the DPP-Ⅳ had a typical β-sheet conformation, and the thermal denaturation temperature was (72.7 ± 0.3) ℃. Metal ions such as Zn²⁺, Fe²⁺ and Fe³⁺ revealed a significant inhibitory effect on the activity of DPP-Ⅳ. The inhibition rate of 1 mmol/L Zn²⁺ on the enzyme’s activity was as high as 98%.