鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

Purification and Characterization of Trypsin Inhibitor from Chickpea Seeds

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法,从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂(CPTI).研究表明:CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%,对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用;用SDS-PAGE测得CPTI近似分子质量为25.7 kD;CPTI具有较高的热稳定性,在100℃下加热60min,对胰蛋白酶活性仍保持78%抑制率;Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型.动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10-7 mol/L.

Chickpea trypsin inhibitor（CPTI） was purified and characterized from the seeds of Cicer arietinum L using ammonium sulfate grade precipitation,anion exchange chromatography（DEAE-cellulose 52）,and gel filtration on Sephadex G-100.The extraction from Cicer arietinum L seeds strongly inhibit trypsin activity by 80%,and inhibit chymotrypsin activity by 32%,but exhibited no effects on three proteases of pepsin,papain and subtilisin.The approximate molecular weight of CPTI was estimated to be 25.7 kD by SDS-PAGE.CPTI was also heat-resistant and maintained 78% of the activity after heated in 100 ℃ for 60 min.The kinetic analysis showed that CPTI inhibited trypsin following the competitive model with an inhibition constant Ki of 3.99 × 10-7 mol /L.