title: Human factor VIII from heparinized plasma. Purification and characterization of a single-chain form.
abstract: Human factor VIII was purified from heparinized blood by cryoprecipitation, poly(ethyleneglycol) precipitation, Affi-Gel blue, aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. A purification of 280,000-fold over plasma with a specific activity over 5300 units/mg was achieved. Analyses of factor VIII using HPLC indicated a molecular mass of 280-340 kDa. Variation in the native mass may reflect heterogeneity of the protein due to associated lipid since structural analysis confirmed that factor VIII contained variable amounts of free fatty acids and diglycerides and triglycerides, but no phospholipids. Additional characterization by denaturing polyacrylamide gel electrophoresis under reducing conditions, followed by silver staining, showed a major single-chain polypeptide of factor VIII with a mass of approximately 260 kDa. To determine whether proteolyzed forms of factor VIII were present during fractionation, we analysed earlier steps in purification. This revealed additional species of factor VIII eluting faster than the single-chain form during chromatography on polyelectrolyte E5. Gel electrophoresis showed that these species of factor VIII consisted of multiple polypeptide chains, and partial peptide mapping using Staphylococcus aureus V8 protease indicated that they were structurally related. Monoclonal and hemophilic antibodies were used in immunoadsorption experiments to demonstrate that the purified factor VIII was composed predominantly of the 260-kDa factor VIII chain.
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005169:Factor VIII / Q000302:isolation & purification* / Q000378:metabolism; D006493:Heparin; D006801:Humans; D007202:Indicators and Reagents; D008970:Molecular Weight; D010449:Peptide Mapping
chemical_list: D007202:Indicators and Reagents; D005169:Factor VIII; D006493:Heparin