Disulfide bonds and the quaternary structure of factor VIII/von Willebrand factor.
Human Factor VIII/von Willebrand factor, purified by calcium citrate-cellulose chromatography and 4% agarose gel filtration was subjected to sodium dodecyl sulfate gel electrophoresis on gels containing 2% acrylamide and 0.5% agarose. We find a series of multimers of which the apparent molecular weight of the higher members was congruent with5 million. The higher multimers were isolated by 2% agarose gel filtration. Treatment of the high molecular weight multimers with 2-mercaptoethanol at concentrations of 0.005-0.5% in the presence of 1% dodecyl sulfate resulted in a shift to lower molecular weight multimers. Between mercaptoethanol concentrations of 0.01 and 0.5%, the predominant species was the dimer of the basic subunit. Mercaptoethanol concentrations >0.5% were required to reduce the interchain disulfide bonds of the dimer. An artificial multimeric series was prepared by cross-linking von Willebrand factor subunits with dimethylsuberimidate. Comparison of the multimers produced by reduction with the multimers produced by cross-linking, demonstrated the absence of odd-numbered multimers from the reduced series. Thus, the protomeric unit appears to be the dimer. High molecular weight multimers had both ristocetin cofactor activity and Factor VIII procoagulant activity. Reduction of the protein in the absence of denaturing agents, caused a gradual shift to lower molecular weight species and a concomitant loss of von Willebrand factor activity. In contrast, Factor VIII activity was unchanged by reduction. These studies suggest that the moieties having von Willebrand factor activity and those having Factor VIII activities are covalently linked by disulfide bonds.
D001779:Blood Coagulation Factors / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides; D005169:Factor VIII / Q000302:isolation & purification; D006801:Humans; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011487:Protein Conformation; D014841:von Willebrand Factor / Q000302:isolation & purification
D001779:Blood Coagulation Factors; D004220:Disulfides; D014841:von Willebrand Factor; D005169:Factor VIII