Perret B A, Furlan M, Beck E A
Biochim Biophys Acta. 1979 May 23;578(1):164-74. doi: 10.1016/0005-2795(79)90124-7.
Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.
通过琼脂糖（Sepharose CL - 2B）凝胶过滤从冷沉淀中分离出人VIII因子相关蛋白。在SDS - 2%聚丙烯酰胺 - 0.5%琼脂糖凝胶上进行电泳，结果显示VIII因子相关蛋白存在大小异质性，这与SDS - 1%琼脂糖凝胶电泳和电子显微镜所显示的相似。将表观分子量与交联IgM寡聚体的表观分子量进行比较，对于在我们凝胶过滤柱空体积附近洗脱的VIII因子，其对应值高达20×10⁶ 。对电泳凝胶上迁移区间的测量表明相邻条带之间存在恒定的大小差异。在Sepharose柱的后续洗脱液中以及用半胱氨酸对VIII因子进行部分还原后，发现了较小的聚集体。为了比较VIII因子相关蛋白大小聚集体之间的大小差异，我们用与辛二亚胺二甲酯交联并随后用2 - 巯基乙醇进行二硫键还原的VIII因子对SDS - 2%聚丙烯酰胺 - 0.5%琼脂糖凝胶进行校准。通过结合校准范围，测量了大小VIII因子聚集体的恒定区间。任何相邻蛋白条带（经免疫鉴定为VIII因子相关蛋白）之间的区间等于基本VIII因子亚基链的二聚体。我们得出结论，VIII因子聚集体对应于二聚体分子的多聚体，即基本亚基链的二聚体对。
