绿豆胰蛋白酶抑制剂的分离和纯化

Separation and purification of mung bean trypsin inhibitor

目的研究1种高效分离纯化绿豆胰蛋白酶抑制剂(MBTI)的方法。方法采用硫酸抽提,硫酸铵分级沉淀,缓冲液溶解沉淀,用相对分子质量(Mr)为30 000的超滤膜去除大分子蛋白质,Mr为5 000的超滤膜除盐及小分子蛋白质,亲和色谱纯化,得MBTI,高效液相色谱法(HPLC)和SDS-PAGE电泳法分别测定其纯度和Mr,以酪蛋白为底物测其活性。结果纯化的MBTI经HPLC和SDS-PAGE鉴定为2条蛋白质带,其Mr分别为12 000和8 000,其活性约为大豆蛋白酶抑制剂的3倍。结论超滤法与亲和色谱结合能快速高效地分离纯化MBTI。

Purpose To extract and to purify the mung bean trypsin inhibitor（MBTI）. Methods The mung bean was prepared by the process of soaking and extracting with the sulfuric acid, precipitating in, saturated ammonium sulfate liquor, and dissolving the precipitation with the 0.05 mol/L pH 4.5 of acetic acid buffer or pH 7.5 of phosphoric acid buffer or double distilled water. Molecular weight is 30 000 ultra filtration membrane doing away with macromolecule protein, and molecular weight is 5 000 ultrafiltration membrane dividends to salt and micromolecule protein. To purify MBTI by affinity chromatography, the purity and molecular weight of mung bean trypsin inhibitor were determined based on HPLC and SDS-PAGE, and the trypsin inhibitory activity was performed by using casein as a substrate. Results The purified MBTI gave two protein bands in HPLC and SDS-PAGE electropboresis. Its Mr was estimated to be 8 000 and 12 000. The MBTI activities were roughly triple than soybean trypsin inhibitor （SBTI）. Conclusion The ultra filtration separation shows the high separation and purification of mung bean trypsin inhibitor.