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The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds.Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in y-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases. Factor IX (Christmas Factor) is the zymogen of a serine protease that participates in the middle phase of the intrinsic pathway of blood coagulation (1). Like several other plasma proteinsi.e., prothrombin, Factor X, Factor VII, Protein C, and Protein S-Factor IX requires vitamin K for its biosynthesis (2-4). Individuals lacking Factor IX (Christmas disease or hemophilia B) show bleeding symptoms essentially identical to those of classic hemophilia or hemophilia A (Factor VIII deficiency) (5, 6). The activity of Factor IX is also depressed in the plasma of patients treated with coumarin analogs, but in such cases the effect can be reversed by the administration of vitamin K (6).Bovine and human Factors IX have been purified and characterized (7)(8)(9)(10)(11)(12) Previous comparison of the partial sequence of bovine Factor IX with the sequences of bovine prothrombin and Factor X revealed homologous regions, indicating that these three proteins may have evolved from a common ancestral protein (16,17). Recently, the light and heavy chains of bovine Protein C, another vitamin K-dependent plasma protein, have been found to be homologous with Factor X (18,19). In the present communication, the complete sequence of Factor IX is presented.Comparison with the sequences of Factor X, Protein C, and prothrombin reveals areas of homology as well as regions lacking obvious similarity. The experimental details of the sequence determination will be published elsewhere. METHODS Factor IX, isolated from bovine plasma (7) and converted to Factor IXaa by a protease from Russell's viper venom (15), was obtained through the courtesy of K. Fujikawa. Factor IXaa was reduced with dithioerythritol and S-alkylated with 4-vinylpyridine. The two peptide chains (segment N and segment C) were separated on a column of SP-Sephadex C-25 by applying a linear gradient of sodium formate buffers in the presence of 7 M urea (20).Segment N (residues 1-181) was divided into two fragments (CB I and CB II), by cleavage with cyanogen bromide under conditions that avoid cleavage at y-carbo...

The amino-acid sequence of the heavy chain of bovine blood coagulation factor X1 (Stuart factor) isolated before and after activation has been determined. Sequence analysis was performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion. Comparison of the complete sequence with those of other hepatic and pancreatic serine proteases demonstrates homology of the heavy chain of activated factor X1 (factor XI.) with the B chain of bovine thrombin as well as with bovine trypsin, chymotrsins A and B, and porcine elastase. The activation peptide cleaved near the amino terminus by a protease from Russell's viper venom differs in both size and sequence from those of other serine proteases. With three exceptions, all of the residues which are important in the catalytic functions of trypsin and chymotrypsin occur in corresponding loci in the heavy chain of factor Xa. These findings suggest that the three-dimensional structure of the heavy chain is similar to that of the pancreatic serine proteases and that these enzymes have evolved from a common ancestral gene.Factor X (Stuart factor) is the zymogen of a protease which upon activation by a complex of activated factor IX (factor IXa) and factor VIII catalyzes the conversion of prothrombin to thrombin (1). Bovine factor X is a glycoprotein with a molecular weight of 55,100. It is composed of two polypeptide chains ("heavy" and "light") held together by one or more disulfide bonds. The intact protein can be separated chromatographically into two components (factor Xi and X2) which are identical in molecular weight and amino-acid composition (2-4). Upon activation by the complex of factors IXa and VIII in the presence of calcium and phospholipid, by factor VII and tissue factor in the presence of calcium, by a protease from Russell's viper venom, or by trypsin, glycopeptides are split, either near the amino or both the amino and carboxyl ends of the heavy chain, and an enzymatically active serine protease, factor Xa, is formed (5-8). The heavy chain of factor Xa is homologous to pancreatic and certain hepatic serine proteases, as evidenced by similarities of the amino-terminal sequence and the sequence surrounding the serine and histidine residues of the active site (9).The sequence of the light chain of bovine factor X1 (10), for which the pancreatic proteases have no counterpart, is homologous to the amino-terminal sequences of bovine prothrombin and factor IX (11). All three of these factors are vitamin K dependent, and this dependency appears to be expressed in the post-transcriptional synthesis of y-carboxyglutamyl residues (12, 13) in the amino-terminal portions of the zymogens.This communication presents the amino-acid sequence of the heavy chain of bovine factor X1, and an examination of the structural homology of this chain with those of other serine proteases. Abbreviation: CHO, carbohydrate. METHODSFactor X1 was isolated from bovine plasma and converted to the active form (factor Xia) by incubation with a protease from Russel...

The detailed proof of the amino acid sequence of the 140 residues (16,193 daltons) of the light chain of bovine factor X1 (Stuart factor) is presented. Sequence analyses were performed on fragments obtained after chemical cleavage of asparagine-glycine and tryptophanyl peptide bonds and after various enzymatic digestion procedures. Twelve gamma-carboxyglutamyl residues are clustered in the amino-terminal 39 residues and 13 half-cystine residues are found in the carboxyl-terminal 91 residues, suggesting two domains in the light chain, one exceptionally anionic and the other extensively cross-linked by disulfides.