延胡索纤溶酶的分离纯化及部分性质

Purification and partial characterization of fibrinolytic enzyme from Rhizoma corydalis

目的 从延胡索（Rhizoma corydalis）中分离纯化具有溶解纤维蛋白活性的单体蛋白单一组分，并对粗酶性质做初步研究。方法利用硫酸铵分级沉淀和阴离子交换色谱从延胡索中纯化纤溶酶。结果从延胡索中纯化出两种纤溶酶，表观相对分子质量分别为33000和59000。粗酶用丝氨酸蛋白酶抑制剂鉴定为丝氨酸蛋白酶，ED-TA对酶活没有抑制作用。延胡索纤溶酶粗酶体外溶纤性质主要表现为激酶活性，在4～50℃活性稳定，50～75℃酶活有所下降；最适pH值为8．0，在pH3、0左右，仍能保留大部分酶活；能抵抗胰蛋白酶水解和胃蛋白酶的作用。结论延胡索纤溶酶性质稳定，有希望研发成为一种溶栓新药。

Purpose To purify the fibrinolytic enzyme from Rhizoma corydalis （yanhusuo） and to analyze its properties. Methods Purifying the enzyme by the ammonium sulfate fractionation and DEAE-Sepharose fast flow chromatography. Results Two components of fibrinolytie enzyme （RCFE-1, RCFE-2）were isolated. The purified enzymes showed a single protein band in the SDS-PAGE. Their relative molecular weights were 33 000 and 59 000 respectively. Crude enzymes were completely inhibited by phenylmethysulfonyl fluoride （PMSF） and not by EDTA, so RCFE was identified as a ser/ne protease. RCFE hydrolyzed fibr/n was mainly by activating plasminogen in vitro. Its activity was stable below 50℃, peaked at pHg. 0, 50-75℃ and around pH 3.0, most activity was still left. Trypsin Could not hydrolyze RCFE by SDS-PAGE. Under stimulating acid environment of stomach, the fibrinolytic activity of crude extract did not decrease. Conclusion Properties of RCFE are stable and are is expected to be a new thrombolytic medicine.