论文部分内容阅读
目的 从圆斑蝰蛇泰国亚种蛇毒中分离纯化凝血 因子 (F )激活剂并研究其部分特性。 方法 采用离子交换柱层析和凝胶过滤等方法进行分离纯化 ,以血液凝固时间及发色底物 (S- 2 2 2 2 )进行活性鉴定和温度、p H值、蛋白酶抑制剂对活性的影响 ,SDS- PAGE进行分子量、等电点及纯度测定 ,苯酚 -硫酸法及间苯二酚 -盐酸法测定糖含量。 结果 所得促凝组分 1 能特异性地活化牛 F ,从而水解 S- 2 2 2 2酰胺键。SDS- PAGE呈现单一蛋白带 ,分子量为 6 1.1± 1.5 k D(还原 ) ,6 0 .4± 1.9k D(非还原 )。等电点为 p H=8.0 0± 0 .0 6。含中性己糖 12 .4 %±0 .5 % ,唾液酸 0 .12 %± 0 .0 2 %。在 5 0℃以下稳定 ,最适 p H为 7.0～ 8.2。可被金属蛋白酶抑制剂 EDTA- Na2 完全抑制 ,二硫苏糖醇 (DTT)及 L -半胱氨酸部分抑制 ,不受胰蛋白酶抑制剂抑肽酶抑制。 结论 从圆斑蝰蛇泰国亚种蛇毒分离纯化的 F 激活剂是一种碱性糖蛋白 ,属于 Ca2 + 依赖性的金属蛋白酶。
Objective To isolate and purify the coagulation factor (F) activator from the snake venom of Asiaticus viridis and to study some of its properties. Methods Separation and purification were performed by ion exchange column chromatography and gel filtration. The activity was identified by blood clotting time and chromogenic substrate (S-222) and temperature, p H value, and protease inhibitor activity. The effect of SDS-PAGE on molecular weight, isoelectric point, and purity was measured. The phenol-sulfuric acid method and resorcinol-hydrochloric acid method were used to determine the sugar content. Results The resulting procoagulant component 1 specifically activates bovine F, thereby hydrolyzing the S-2222 amide bond. SDS-PAGE showed a single protein band with a molecular weight of 6 1.1±1.5 kD (reduced) and 60.4±1.9 kD (non-reduced). The isoelectric point is p H =8.0 0 ± 0 . 0 6. Neutral hexose with 12.4% ± 0.5%, sialic acid 0.12% ± 0.02%. It is stable below 50C, and the optimum pH is 7.0-8.2. Can be completely inhibited by the metalloproteinase inhibitor EDTA-Na2, dithiothreitol (DTT) and L-cysteine partial inhibition, not inhibited by the trypsin inhibitor aprotinin. Conclusion The F activator isolated and purified from the subspecies of Thailand snake venom was a basic glycoprotein belonging to Ca2+-dependent metalloproteinases.