Radcliffe R, Nemerson Y
J Biol Chem. 1975 Jan 25;250(2):388-95.
Factor VII purified as previously described, was found to consist of two polypeptide chains joined by disulfide bridges. We now report the isolation and 200,000-fold purification of a single chain form of Factor VII. This was accomplished by protecting the molecule against proteolysis by including benzamidine during the entire purification. The purification was essentially as previously reported except that barium cirtate was substituted for barium sulfate as an absorbant for Factor VII as it resulted in a 4-fold increase in yield. Single chain Factor VII is rapidly hydrolyzed by Factor Xa in the presence of calcium ions and phospholipids, and by thrombin, to a two-chain form which possesses at least 85 times the Factor VII clotting activity of the single chain species. The two-chain form of the enzyme requires tissue factor in order to activate Factor X. From the observed rates of activation of Factor VII by Xa in the presence of calcium ions and phospholipids, it was calculated that at approximately physiological concentration, Factor VII activity would increase at an initial rate of 20-fold per min; this reaction is sufficiently rapid to constitute a feedback control mechanism. The action of thrombin is approximately 40-fold slower under these conditions. Diisopropylphosphorofluoridate inactivates the single chain and two-chain forms of Factor VII at approximately equal rates. After inhibition, the single chain species could be cleaved but not activated by proteolysis.
如前所述纯化的凝血因子VII由通过二硫键连接的两条多肽链组成。我们现在报告一种单链形式凝血因子VII的分离及20万倍纯化。这是通过在整个纯化过程中加入苯甲脒保护分子不被蛋白水解来实现的。纯化过程基本上与之前报道的相同，只是用柠檬酸钡替代硫酸钡作为凝血因子VII的吸附剂，因为这使产量提高了4倍。单链凝血因子VII在钙离子和磷脂存在下会被因子Xa迅速水解，并被凝血酶水解为双链形式，其凝血因子VII的凝血活性至少是单链形式的85倍。该酶的双链形式需要组织因子才能激活因子X。根据在钙离子和磷脂存在下因子Xa激活凝血因子VII的观察速率计算，在大约生理浓度下，凝血因子VII活性将以每分钟20倍的初始速率增加；该反应足够迅速，可构成一种反馈控制机制。在这些条件下，凝血酶的作用大约慢40倍。二异丙基氟磷酸酯以大致相同的速率使凝血因子VII的单链和双链形式失活。抑制后，单链形式可被蛋白水解切割但不能被激活。
