人尿激肽原酶的纯化与鉴定

采用两性离子胶体沉淀和乙醇沉淀相结合的粗提方法，经离子交换、疏水层析、亲和层析及凝胶过滤4个步骤有效地将人尿激肽原酶（hk-1）粗提物纯化，比活提高了1 755倍，总得率为70%.用以慈菇蛋白酶抑制剂为配体的亲和层析纯化hk-1，效果理想，整个工艺路线适合产业化生产.纯化产物在SDS-聚丙烯酰胺凝胶电泳上为单带,高压液相色谱(HPLC)上为单峰,基质辅助激光解析电离飞行时间质谱测得分子质量为33 450 u，等电聚焦测得pI在4.3附近，为含糖蛋白.同时测定了该酶的热稳定性和pH稳定性.纯化过程中同时分离得到另一种药用蛋白——人尿胰蛋白酶抑制剂（HUTI）.

The Purification and Identification of Human Urinary Kallikrein

Human urinary kallikrein (hk-1) and human urinary trypsin inhibitor (hUTI) are two acidic proteins in the urine. Both of them are important drugs. The way of colloid adsorption together with ethyl alcohol precipitation isolated these two proteins successfully. By using of ion-exchange, hydrophobic, affinity chromatography and gel filtration, human urinary kallikrein(hk-1) was purified with single band on SDS-PAGE and single peak on HPLC. The molecular mass was 33 450 u on MALDI-TOF-MS, the pI was about 4.3 on IEF. The influence of pH and temperature on the activity of hk-1 was studied. The whole procedure is suitable for large-scale production.