Preparation and properties of bovine factor VIII (antihemophilic factor).
Preparation and properties of bovine factor VIII (antihemophilic factor).
复制标题
牛因子VIII（抗血友病因子）的制备和性质。
作者：
G. Vehar;E. Davie
通过硫酸铵分级分离、甘氨酸沉淀、DEAE-Sephadex柱层析、硫酸盐-Sepharose柱层析、SephadexG-200凝胶过滤和因子X-Sepharose柱层析，从牛血浆中纯化因子VIII约300000倍。高度纯化的制剂迁移作为一个三重十二烷基硫酸钠/尿素-聚丙烯酰胺凝胶电泳的表观分子量为93000，88000，和85000。纯化制剂的凝血活性被兔体内产生的抗纯化因子VIII蛋白或因子VIII/von Willebrand因子制剂的抗体抑制。纯化蛋白的抗体也抑制凝血因子VIII/血管性血友病因子制剂的凝血活性。纯化的因子VIII不含血小板聚集活性，在人富血小板血浆中测得。在因子IXa、钙和磷脂存在的情况下，因子X的活化需要因子VIII的纯化制剂。它被激活约30倍凝血酶或因子Xa加钙和磷脂，这些反应中的每一个都伴随着十二烷基硫酸钠/尿素-聚丙烯酰胺凝胶电泳模式的蛋白质的变化。在需要钙和磷脂的反应中，因子VIII被牛活化蛋白C迅速灭活。该反应也与高度纯化的蛋白质的十二烷基硫酸钠/尿素-聚丙烯酰胺凝胶电泳模式的变化有关。涉及三种高度特异性丝氨酸蛋白酶的这些实验支持在聚丙烯酰胺凝胶上观察到的三联体是因子VIII的结论。
Factor VIII has been purified approximately 300000-fold from bovine plasma by ammonium sulfate fractionation, glycine precipitation, DEAE-Sephadex column chromatography, sulfate--Sepharose column chromatography, Sephadex G-200 gel filtration, and factor X--Sepharose column chromatography. The highly purified preparation migrated as a triplet on sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis with apparent molecular weights of 93000, 88000, and 85000. The coagulant activity of the purified preparations was inhibited by antibodies raised in rabbits against either the purified factor VIII protein or a preparation of factor VIII/von Willebrand factor. Antibodies to the purified protein also inhibited the coagulant activity of factor VIII/von Willebrand factor preparations. The purified factor VIII contained no platelet-aggregating activity, as measured in human platelet-rich plasma. The purified preparation of factor VIII was required for the activation of factor X in the presence of factor IXa, calcium, and phospholipid. It was activated about 30-fold by thrombin or factor Xa plus calcium and phospholipid, and each of these reactions was accompanied by a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the protein. Factor VIII was rapidly inactivated by bovine-activated protein C in a reaction requiring calcium and phospholipid. This reaction was also associated with a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the highly purified protein. These experiments involving three highly specific serine proteases support the conclusion that the triplet observed on polyacrylamide gels is factor VIII.