Perret B A, Furlan M, Kneubuehl F, Beck E A
Biochim Biophys Acta. 1981 Jun 29;669(1):98-104. doi: 10.1016/0005-2795(81)90228-2.
We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.
我们设计了一种电泳系统，用于分离具有生物活性的单个凝血因子VIII多聚体。通过在琼脂糖CL - 2B上进行凝胶过滤从血浆冷沉淀中纯化得到的人凝血因子VIII，在不含十二烷基硫酸钠（SDS）的情况下，于pH 7.5的0.04 M Tris/0.06 M Tes缓冲液中的2.0%聚丙烯酰胺凝胶上进行电泳。用考马斯亮蓝染色显示出一系列蛋白条带。电泳迁移率的测量表明相邻条带之间的大小间隔恒定。在存在SDS的情况下进行二维电泳，得到了相同的迁移率顺序，这表明在第一个电泳系统中凝血因子VIII蛋白不同的迁移速率是由大小决定的，而非电荷决定。在不存在SDS的情况下进行电泳后，通过从凝胶切片上洗脱，回收了凝血因子VIII促凝活性、瑞斯托霉素辅因子活性以及凝血因子VIII相关抗原。活性峰的分布类似于在对照凝胶中发现的考马斯亮蓝染色的凝血因子VIII蛋白的分布。因此，我们证明即使在保留凝血因子VIII活性的中性pH条件下，对凝血因子VIII多聚体进行电泳分离也是可行的。
