尖吻蝮蛇毒凝血样酶的纯化、性质及其萤光光谱

PURIFICATION, PROPERTIES AND FLUORESCENCE SPECTRA OF THE THROMBIN-LIKE ENZYME OF AGKISTRODON ACUTUS VENOM

徐洵等用DEAE-Sephadex A-50将尖吻蝮蛇毒分离成十二个组份。经证明第十组份有凝血样酶活性。将此组份经Sehadex G-75、DE52柱进一步分离得到没有完全分开但都有凝血样酶活性的两个峰。用聚丙烯酰胺凝胶电泳检查,均为一条带。SDS凝胶电泳测分子量都在30,000左右,免疫扩散图谱显示二者不形成交叉沉淀线,说明这二个组份是很不专一的凝血样酶。最适pH为7.4,等电点接近4。 研究组份Ⅱ的自身萤光光谱,在中性缓冲液中与牛凝血酶自身萤光光谱比较,有所不同。表明这两个酶在构象上是有差异的。凝血样酶在pH2—9范围内与色氨酸比较,萤光光谱峰没有位移,活性变化伴随着萤光强度强弱变化。变温时凝血样酶萤光强度曲线与色氨酸变温曲线接近。凝血样酶活性不被肝素抑制,表现在其萤光强度基本不变。

Xu Xun ct. al. have separated A. acutus venom into 12 fraction using DEAE-sephadex A-50. The 10th fraction has been proved to possess the activity of thrombin-like. The further separation of the fraction into two bands which, though not well resolved, both have the activity of thrombin-like was achieved by gel filtration on Sephadex G-75 or DE-52. Potyacrylamide gel electrophoresis indicated that both of them were homogeneous.. They have a molecular weight of about 30,000 as estimated from the electrophoresis on SDS-gel. Their immunodiffusion spectrum showed no cross-line of precipitation, indicating their properties were similar to each other.Their optimum pH and isoelectric point were at 7.4 and near 4 respectively. We also studied the intrinsic fluorescence spectrum of fraction 2. Its spectra in neutral buffer was different from that of bovine thrombin, suggesting the conformational difference between pH 2 - 9 showed no peak shift in reference to that of Trp. Its activity varied with the change in the strength of fluorescence. The curve of the strength of fluorescence of thrombin-like ploted against the temperature resembled that of Trp. The inscnsitiveness of the strength of the fluorescence of thrombin-likc to the addition of heparin demonstrated that the activity of thrombin-like can not be inhibited by heparitu.