Affinity Chromatography of Factor VIII Subunits
Gel filtration of factor VIII resulted in elution of a fraction which possessed procoagulant activity (VIII:C), ristocetin cofactor (VIIIrRCF) and antigenic activity (VIII:Rag). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of this preparation showed that no protein entered the gel. Eight bands ranging from 30,000 to 230,000 molecular weight (mw) were seen when 400μg reduced protein was loaded onto the SDS-PA geL Attempts to assay the functional properties were made in order to investigate whether the multiband structure was a property of factor VIII or due to contaminants. These proved unsuccessful, as reduction and SDS-PAGE of factor VIII destroyed its functional properties. Affinity chromatography with human and rabbit antibodies to factor VIII provided an alternative method of relating the subunit structure to functional properties. Insolubilized human antibody inactivated VIII:C and did not bind VIII:Rag. The protein bound to human antibody was dissociated with NH SCN and reduced. SDS-PAGE showed heavy protein staining, with 95% of the protein present in 35,000 and 62,000 mw bands. Similar treatment of a control human IgG column showed minimal protein in the same mw region. Using insolubilized rabbit antibody to human factor VIII, all 8 bands were observed after N^SCN dissociation, reduction and SDS-PAGE. These results suggest that contaminat^ely that the low bands observed on heavily loaded gels are due to protein contamination.