Immunoaffinity purification of bovine factor VII
Immunoaffinity purification of bovine factor VII
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牛VII因子的免疫亲和纯化
DOI：
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发表时间：
1984
期刊：
影响因子：
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通讯作者：
Y. Nemerson
中科院分区：
文献类型：
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作者：
R. Bach;J. Oberdick;Y. Nemerson
因子VII已通过包括在免疫吸附柱上亲和纯化的程序从牛血浆中纯化至均一。使用3 H-因子VII作为内标，通过凝血试验和液体闪烁计数测定回收率。通过两种方法计算的纯化因子约为120，000倍，最终产率约为18%。通过十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳评估均一性。该物质以53，000道尔顿的单链多肽链迁移，在被因子Xa激活后，单链酶原定量转化为双链因子VIIa。将亲和纯化的因子VII转化为因子VIIa导致凝血活性的高达119倍的活化，这比报道的通过其他方法制备的因子VII的活化性大2.7-4倍。Zur等人计算出，未被痕量因子VIIa污染的纯因子VII在转化为因子VIIa时将被激活123倍。观察到的亲和纯化的因子VII的活化性和理论预测之间的密切协议表明，我们已经分离出因子VII基本上没有因子VIIa。三批牛血浆的纯化数据得出因子VII的血浆浓度估计值为10.1 nM至18.5 nM。
Factor VII has been purified to homogeneity from bovine plasma by a procedure that includes affinity purification on an immunoadsorbent column. Recovery was determined by both coagulant assay and liquid scintillation counting, using 3H-factor VII as an internal standard. The purification factor calculated by both methods was approximately 120,000-fold, with a final yield of approximately 18%. Homogeneity was assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The material migrated as a single polypeptide chain of 53,000 daltons, and following activation by factor Xa, the one-chain zymogen was quantitatively converted to two-chain factor VIIa. Conversion of affinity-purified factor VII to factor VIIa resulted in up to a 119-fold activation of the coagulant activity, which is 2.7–4 times greater than the activatability reported for factor VII prepared by other methods. Zur et al. calculated that pure factor VII, uncontaminated by traces of factor VIIa, would be activated 123-fold upon conversion to factor VIIa. The close agreement between observed activatability of affinity-purified factor VII and the theoretical prediction suggests that we have isolated factor VII essentially free of factor VIIa. The purification data from three lots of bovine plasma yield an estimate for the plasma concentration of factor VII from 10.1 nM to 18.5 nM.