Human coagulation factor IX was purified by two ion-exchange chromatographies on DEAESephadex A-50, heparin-Sepharose chromatography, hydroxyapatite chromatography and immunoadsorbent 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 s20, = 3.97 S. A partial specific volume of V = 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. contained approximately 17.5 % carbohydrate, which includes 4.7 % hexose, 6.8 amine and 6 % sialic acid.points of the major components lay within the range of pH 4.0 to 4.6. dependent coagulation factors measured by coagulation factor assays or immunodiffusion in gels.cm2 s-' and a frictional ratio fife = 1.54.Coagukation factor IX is a protein which takes part in the intrinsic coagulation pathway of blood coagulation. The activated factor IX (F IXa) forms a complex with factor VIII and Ca" which then activates factor X to factor Xa. The exact activation mechanism and the role of factor IXa in the complex is not fully understood [l]. A prerequisite for the demonstration of the exact role of factor IX in blood coagulation is the availability of pure factor IX.Bovine factor IX has been recently purified and the molecular characteristics described [2].The purification of human factor IX has been studied extensively during recent years. Abbreviations. IgG, immunoglobulin G ; dansyl, 5-dimethylaminonaphthalene-1 -sulphonyl.Enzyme. Prothrombinase or factor Xa (EC 3.4.21.6).ing BaS04 adsorption, DEAE-cellulose chromatography, preparative electrophoresis and immunosorption, and the work of Anderson et al.[5] using ion exchange, heparin-Sepharose chromatography and gel filtration have been published. Partial characterization of factor IX was described in both of those papers. In a previous publication [6] I have described a method for the isolation and some molecular properties of human factor IX. In this paper an improved purification procedure for human factor IX is described together with its chemical, physico-chemical and immunological properties.
MATERIALS AND METHODS
ChemicalsFactor IX concentrate [7] and human albumin were prepared in this laboratory. Bovine thyroglobulin (type l), heparin (grade l), ovalbumin (grade V) and bovine serum albumin (fraction V) were obtained from