Isolation and Characterization of Human Factor VIII (Antihemophilic Factor)

Abstract

Factor VIII (antihemophilic factor) has been purified approximately 500-fold from the cryoprecipitate fraction of human plasma. The isolation procedure involves adsorption of contaminants with A1(OH)3, column chromatography on tricalcium citrate-cellulose, precipitation with concanavalin A, and an agarose gel filtration step. The final product is homogeneous when examined by zone electrophoresis, sedimentation equilibrium, and immunoelectrophoresis. The molecular weight determined by sedimentation equilibrium is 1.12 million ± 98,000. After reduction of the protein with 2-mercaptoethanol or dithiothreitol, subunits are formed which migrate as one band in polyacrylamide gel electrophoresis and zone electrophoresis. The subunits are heterogeneous, however, in the ultracentrifuge, apparently due to substantial aggregation. The smallest species which could be detected has a molecular weight of 105,000 ± 5,000. The molecular weight of the subunit determined by sodium dodecyl sulfate (SDS) gel electrophoresis was 240,000. The latter value may be high, however, due to the fact that human Factor VIII contains approximately 6% carbohydrate (hexose, hexosamine, and neuraminic acid) and the molecular weights of glycoproteins determined by SDS gel electrophoresis tend to be high. Antibodies prepared in rabbits against human Factor VIII inhibit both human and bovine Factor VIII activity. Antibodies to the highly purified human Factor VIII also form a precipitin line in immunoelectrophoresis experiments with the cryoprecipitate fraction prepared from hemophilic plasma, indicating that an abnormal Factor VIII molecule is present in the plasma of individuals with classic hemophilia. Other general properties of human Factor VIII, including its amino acid composition, thrombin modification, and turnover in hemophilic dogs, are also reported.