Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 16-19, January 1975 Bovine Factor X1 (Stuart Factor). Primary Structure of the Light Chain (amino-acid sequence/blood coagulation) DAVID L. ENFIELD, LOWELL H. ERICSSON, KENNETH A. WALSH, HANS NEURATH, AND KOITI TITANI Department of Biochemistry, University of Washington, Seattle, Wash. 98195 Contributed by Hans Neurath, October 21, 1974 ABSTRACT The amino-acid sequence of the light and on five large fragments prepared as shown diagrammati- chain of bovine factor X1 is presented. The sequence of 112 cally in Fig. 1. Two fragments were obtained by cleavage of of the 140 residues was determined automatically on fragments produced by specific cleavage of arginyl, glu- asparaginyl-glycine bonds (10) and one fragment each by tamyl, tryptophanyl, and asparaginyl-glycine bonds. The cleavage of peptide bonds adjacent to tryptophanyl (11) and remainder was determined by conventional procedures. glutamyl (12) residues. The fifth fragment was isolated after a The amino-terminal sequence of the light chain is homol- cleavage adjacent to arginyl residues, requiring succinylation ogous with the amino-terminal region of bovine prothrom- and blocking of free carboxyl groups bin and, like the latter, appears to contain several residues of E-amino groups (13) of a recently discovered unusual amino acid, -y-carboxy- with glycinamide (14, 15). These sequenator analyses pro- glutamic acid. The role of this amino acid in the calcium- vided the sequence of 112 residues. The sequences of the re- binding ability of factor X and prothrombin is discussed. maining residues were determined by conventional Edman degradation techniques (16, 17), using tryptic, chymotryptic, Bovine factor X (Stuart factor) is the zymogen of a protease -and by digestion with activation is me- peptic, and thermolytic peptides, involved in blood coagulation. Zymogen carboxypeptidases A and B. diated by activated factor IX (factor IXa), in the presence of Amino-acid analyses were performed on Beckman (model factor VIII, calcium, and phospholipid, as well as by Russell's 120C) and Durrum (model D-500) amino-acid analyzers. viper venom and by trypsin (1-3). Activated factor X (factor as (13). thrombin. Half-cystine was determined S-pyridylethylcysteine Xa) catalyzes the conversion of prothrombin to Amide sidecha1*s were identified after digestion with amino- Purified bovine factor X can be separated chromatograph- electrophoresis at pH similar peptidase M (18' or by high voltage ically into two fractions (factors XI and X2) having 6.5. chemical and biological properties (4, 5). As commonly de- scribed, factor X is a glycoprotein of molecular weight 54,000, RESULTS containing about 10% carbohydrate and composed of two The amino-acid sequence of the light chain of bovine factor polypeptide chains linked by one or more disulfide bonds Xi is shown in Fig. 2. The amino-acid compositions, as deter- (4, 6). The light and heavy chains have molecular weights of mined by analysis and as calculated from the sequence, are 16,000 and 38,000, respectively; the carbohydrate is exclu- compared in Table 1. The agreement is good except for a dis- sively associated with the heavy chain. crepancy in aspartic acid content. The heavy chains of bovine factors X5 and IXa are homol- Sequenator analysis A (Fig. 1) yielded the first 40 amino- ogous with trypsin, thrombin, and other mammalian serine terminal residues of the intact protein, except for residues 32, proteases (7, 8). The homology is particularly apparent in the 35, 36, and 39. The aspartic acid at position 38 must be con- amino-terminal regions and in the peptide segments sur- sidered tentative because considerable overlap from the pre- rounding the serine residue of the active site. Furthermore, vious step normally occurs after multiple consecutive deg- the amino-terminal sequences of the zymogens, factor IX and radations, and one of the peaks characteristic of threonine on prothrombin, and of the light chain of factor X are also the gas chromatograph coincides with that representing homologous (9). These homologies suggest that these three aspartic acid (19). In addition, all of the glutamic-acid resi- serine proteases involved in blood coagulation and the pan- dues identified within the first 30 amino-terminal residues creatic serine proteases have evolved from a common an- were observed at lower levels than expected. This was espe- cestral gene. For this reason, and in order to establish a basis cially striking at positions 6 and 7, but became less marked in for relating the chemical structure of factor Xa to its func- subsequent degradation steps. tion, the determination of its amino-acid sequence was under- When the light chain was subjected to chymotryptic di- taken. This communication presents a preliminary account gestion, a large "core" peptide derived from the amino- of the sequence of the light chain. terminal region of the molecule remained undigested and could be isolated by precipitation at pH 4. (The term "core" is METHODS intended to imply nothing more than resistance to proteol- Purified bovine factor X, was isolated from bovine plasma (4) ysis.) The amino-terminal sequence of the "core" is Alar- by Dr. Kazuo Fujikawa. The protein was reduced and py- Asn-Ser-Phe, and its amino-acid composition (Table 1) cor- ridylethylated, and the two chains were separated as de- responds to residues 1-44 in the sequence, if residues 32, 35, scribed (4). 36, and 39 are each either glutamic acid or glutamine. Anal- Automatic sequence analysis was performed with the ysis B, beginning with ?-Vala0-Phe-?, was extended to 12 turns Beckman Sequencer (model 890B) on the intact light chain on a fragment obtained by specific tryptic cleavage of arginyl 16 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Primary Structure of Factor X1 17 Arg Asn A Arg Trp Glu Asn 5 10 15 V777777=77-T.~l 28 41 51 63 86 93 Ala Asn Ser Phe Leu Glut Glut Val Lys Gin Gly Asn Leu Glut Arg Leu Giu* Ala Val SOURCES OF FRAGMENTS 16 Glu' Cys Leu Glut Glu- Ala Cys Ser Glut Arg Glut 31 Phe Glu* Asp Ala Glu* Gin Thr Asp* Gin' Phe Trp Ser Lys Tyr Lys I. Cleavage at Arginines 28 and 86 after Carboxyl Glu His Pro Leu Asn Gin Gly His Group Modification. 46 Asp Gly Asp Gin Cys Gly Cys Thr Thr Ala Glu B 61 Cys Lys Asn Gly Ile Gly Asp Tyr Cys Cys Gly 76 Phe Glu Gly Lys Asn Cys Glu Phe Ser Thr Arg Glu Ile Cys Ser 91 Leu Asp Asn Gly Gly Cys Asp Gin Phe Cys Arg Glu Glu Arg Ser II. Cleavage at Tryptophan 41 106 Glu Val Arg Cys Ser Cys Ala His Gly Tyr Val Leu Gly Asp Asp C 121 Ser Lys Ser Cys Val Ser Thr Glu Arg Phe Pro Cys Gly Lys Phe 136 Thr Gin Gly Arg Ser FIG. 2. The amino-acid sequence of the light chain of bovine l. Cleavage at Glutamic Acid 51 factor Xi. Asterisks (*) indicate tentative identifications (see D text). Hyphens between residues have been omitted. JZ. Cleavage at Asporaginyl-Glycines 63-64and 93-94 fragment beginning with the amino-terminal sequence of the E F light chain and two additional fragments. The smaller of these yielded the sequence of 25 residues (analysis E) beginning with SUMMARY OF SEQUENATOR ANALYSES Gly64-le-Gly-Asp, which placed this fragment by overlap A with analysis D. The other fragment, consisting of the car- B boxyl portion of the light chain, yielded the sequence of 29 residues (analysis F) beginning with Gly94-Gly-Cys-Asp. The E sequences of residues 89-94 and 122-140 were completed by F conventional techniques on tryptic, chymotryptic, and ther- molytic peptides. FIG. 1. Diagrammatic summary of fragments generated from the light chain of factor XI for sequenator analysis. The top bar TABLE 1. Amino-acid composition of the light chain represents the light chain and the residues that are important for of bovine factor Xi and of the chymotryptic and peptic its fragmentation. The capital letters, A-F, identify the sequen- "core" peptides* ator analyses in the order in which they are described in the text. The hatched section of each horizontal bar indicates the segment Found Chymo- of sequence determined by that analysis. The overlaps among Light in tryptic Peptic these segments are illustrated by the arrows in the lower portion Amino acid chaint sequence "core"$ "core"§ of the figure. Aspartic acid 14 15 4 4 bonds after all free carboxyl groups were modified with gly- Threonine 6 6 1 1 cinamide. The phenylthiohydantoins of the aspartic and Serine 11 11 3 2 glutamic-acid residues modified in this manner could not be Glutamic acid 27 27 14 14 identified in the gas chromatograph, but, assuming that all Proline 2 2 14 1 1 blank positions involve acidic residues, the results are con- Glycine 14 6 6 4 4 sequence given in Fig. 2. This region of the Alanine sistent with the 2 of the Half-cystine 15 15 2 sequence is being investigated further. Subdigestion Valine 5 5 2 2 chymotryptic "core" with pepsin yielded another "core," cor- Isoleucine 2 2 responding in composition to residues 1-40 (see Table 1), as Leucine 7 7 4 4 well as free tryptophan and two small peptides, i.e., Trp41- Tyrosine 3 3 1 Ser-Lys-Tyr and Ser42-Lys-Tyr. These results established the Phenylalanine 8 8 3 3 carboxyl-terminal sequence of the chymotryptic "core" pep- Lysine 7 7 2 1 tide. Histidine 3 3 In order to extend the sequence beyond the "core" region, Arginine 8 8 2 2 cleavage adjacent to the tryptophanyl residue was used.