苦荞胰蛋白酶抑制剂的纯化及特性研究

利用固相胰蛋白酶亲和色谱及离子交换色谱,从苦荞种子中分离纯化了三个具有抑制胰蛋白酶活力的组份(BTI-Ⅰ,Ⅱ,Ⅲ)。经聚丙稀酰胺凝胶等电聚焦测定,三种抑制剂的等电点为6.87,6.7,5.14。根据Sephadex G-75凝胶过滤,SDS-PAGE测定,以及由当量抑制比值和氨基酸组份的推算,它们的分子量范围分别为5200-5700(Ⅰ),5500—6000(Ⅱ),5000—5600(III)。三者对胰蛋白酶的抑制常数分别为2.12×10~(-8),4.78×10~(-9),1.22×10~(-8)。氨基酸分析结果表明,它们均含有很高的极性氨基酸。在酸性条件下,它们均具有很高的热稳定性。化学修饰的结果表明,它们活性中心的氨基酸残基为Lys。

Purification and Properties of the Inhibitors from Buckwheat Seed

Three components (BTI-Ⅰ , Ⅱ, Ⅲ) of the trypsin inhibitors in buckwheat (Fagopyrum tataricum) seeds were isolated and purified by affinity chromatography with immobilizied trypsin and by ion exchange chromatography on CM-cellu-lose-52 and DEAE-cellulose-52. Isoelectric point of them was 6.87, 6.7 and 5.14 respectively as measurep by PAGE-isoelectric focusing.The molecular weight of each fractions Ⅰ , Ⅱ , Ⅲ was estimated to be 5200-5700, 5500-6000 and 5000-5600 respectively by Sephadex G-75 gel filatration, SDS-PAGE electrophoresis, weight ratio between inhibitor and trypsin at stoichiometric inhibition and the data of amino acid analyses. Amino acid compositions of these fractions revealed that high contents of polar amino acids were present. These inhibitors were highly heat stable under the acid condition. The chemical modification of these inhibitors showed that Lys. residue is essential in their active sites