Human Coagulation Factor IX

Abstract

Human coagulation factor IX was purified by two ion-exchange chromatographies on DEAE-Sephadex A-50, heparin-Sepharose chromatography, hydroxyapatite chromatography and immuno-adsorbent technique. Factor IX was homogeneous by ordinary and sodium dodecylsulphate disc electrophoresis, N-terminal amino acid analyses and ultracentrifugation and by immunological criteria. The following molecular data were observed: 1 Sedimentation equilibrium indicated a molecular weight of 66 100 and sedimentation velocity gave S_{20, w}= 3.97 S. A partial specific volume of v̄= 0.712 ml/g was calculated from the amino acid and carbohydrate composition. 2 Sodium dodecylsulphate disc gel electrophoresis suggested a molecular weight of 65 000. 3 Gel filtration indicated a Stokes radius of 4.08 nm, and ‘a molecular weight’ of 72 000, as well as a diffusion coefficient D_{20, w}= 5.15 × 10⁻⁷ cm² s⁻¹ and a frictional ratio f/f₀= 1.54. 4 Tyrosine was the N-terminal amino acid. The amino acid composition is described. Factor IX contained approximately 17.5% carbohydrate, which includes 4.7% hexose, 6.8%N-acetylhexos-amine and 6% sialic acid. 5 Microheterogeneity of pure factor IX was demonstrated by isoelectric focusing. The isoelectric points of the major components lay within the range of pH 4.0 to 4.6. 6 The antibody raised in rabbits against the pure factor IX did not react with the other vitamin-K-dependent coagulation factors measured by coagulation factor assays or immunodiffusion in gels.