牛源二肽基肽酶-IV的高效制备及性质

Purification and Characterization of Dipeptidyl Peptidase-IV from Bovine Kidney

以高效制备二肽基肽酶IV(dipeptidyl peptidase-IV,DPP-IV)为目的,以牛肾为原料,经酸沉淀、硫酸铵盐析和凝胶过滤层析获得高纯度DPP-IV。还原条件下,纯化蛋白的分子质量为106 kDa,纯化倍数为169.9,得率为9.5%。对该蛋白进行质谱鉴定得到12个肽段,共517个氨基酸残基,与牛DPP-IV氨基酸序列的匹配度为100%。对其性质研究发现,DPP-IV为糖蛋白且以二聚体形式(210 kDa)存在,等电点为5.8。DPP-IV的二级结构具有典型的β-折叠构象,热变性温度为(72.7±0.3)℃。金属离子Zn^(2+)、Fe^(2+)及Fe^(3+)对DPP-IV活力有明显的抑制作用,1 mmol/L的Zn^(2+)对DPP-IV力抑制率高达98%。

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 169.9and 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 typicalβ-sheet conformation,and the thermal denaturation temperature was(72.7±0.3)℃.Metal ions such as Zn^(2+),Fe^(2+)and Fe^(3+)revealed a significant inhibitory effect on the activity of DPP-IV.The inhibition rate of 1 mmol/L Zn^(2+)on the enzyme’s activity was as high as 98%.