/. Blochem. 97, (1985)

Transcription

1 /. Blochem. 97, (1985) A Method for Systematic Purification from Bovine Plasma of Six Vitamin K-Dependent Coagulation Factors: Prothrombin, Factor X, Factor IX, Protein S, Protein C, and Protein Z 1 Nobukazu HASHIMOTO, 1 Takashi MORITA,' and Sadaaki IWANAGA Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, November 5, 1984 A systematic purification scheme is presented for the isolation of six vitamin K- dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B). Prothrombin, factor X, factor IX, and factor VII are well-known plasma serine protease zymogens, all of which are major components associated closely with the blood coagulation pathway (/). All the factors have now been isolated and their whole amino acid sequences, except for that of factor VII, have been established (2). Recently, three additional vitamin K-dependent proteins, designated as protein C (3), protein S (4), and protein Z (5), have been isolated and their physio- 1 This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. 1 Current address: Nihon Medi-Physics Co., Ltd., Takarazuka, Hyogo 665. To whom correspondence should be addressed. Abbreviations: MCA, 4-methylcoumaryl-7-amide; Boc, N-rm-butoxycarbonyl, SDS, sodium dodecyl sulfate; DFP, di/sopropyl phosphorofluoridate; Gla, jmarboxyglutamic acid; PT, prothrombin; PAGE, polyacrylamide gel electrophoresis. Vol. 97, No. 5,

2 1348 N. HASHIMOTO, T. MORITA, and S. IWANAGA logical functions and whole primary structures are being studied. Over the past decade, efforts have focused mainly upon the structure-function relationships of these clotting factors (2). However, it now seems important to investigate the functional role of individual clotting factors in the reaction sequence of the blood coagulation cascade, using a reconstitution system in the presence of accessory components, such as Ca >+, phospholipids and protein cofactors. As a prerequisite for these studies, simpler and more efficient methods for the preparation of milligram quantities of functionally active and biochemically well-characterized clotting factors are essential. The purpose of this work was to establish a purification method for multiple vitamin K-dependent proteins from a single pool of bovine plasma. Although several methods have been elaborated for the preparation of one or at most two pure clotting factors in fair yield (7, 2), there is no protocol for the systematic purification of multiple components with high resolution and high yield from a single pool of plasma. We describe here our procedures for purifying six vitamin K-dependent proteins and demonstrate the usefulness of dye ligand chromatography for the separation of protein C and protein Z. MATERIALS AND METHODS Materials DEAE-Sephadex A-50 and Blue- Sepharose CL-6B were products from Pharmacia Fine Chemicals, Uppsala. Boc-Leu-Ser-Thr-Arg- MCA was purchased from the Protein Research Foundation, Minoh, Osaka. DFP was from Katayama Chemical Co., Osaka. Heparin (152 units/ mg) was a product of Nakarai Chemical Ltd., Kyoto. y-carboxyglutamic acid (Bachem. AG, Switzerland) was a kind gift from Dr. J. Stenflo, Department of Clinical Chemistry, University of Lund, Malmo, Sweden. All other chemicals were of the highest quality available. Hepann-Sepharose CL-6B was prepared by the method of March et al. (6). Benzamidine-CH-Sepharose was prepared by coupling CH-Sepharose (Pharmacia Fine Chemicals) with /vaminobenzamidine in the presence of l-ethyl-3-(3-dimethylamino propyl)carbodiimide (Protein Research Foundation) (7). Bovine a-thrombin (3,500 NTH units/mg, 93 % titrable by /i-nitrophenyl />'-guanidinobenzoate) was isolated by benzamidine-ch-sepharose and SP-Sephadex C-50 chromatographies from the activation mixture of bovine prothrombin activated with bovine factor Xa (#). The factor X-activating coagulant protein (RW-XCP) that activates factor IX was purified to homogeneity from Russell's viper venom by the method of Kisiel et al. (9). Insoluble RW-XCP beads were made by coupling purified RW-XCP with Affigel 10 (Bio-Rad Laboratories, Richmond, California). Factor TKafl was prepared from factor LX by converting factor LX to factor IXa/S with insoluble RW-XCP at 37 C for 2 h in the presence of 10 mm CaQj. Factor LXa/? was purified to homogeneity by gel filtration on Sephacryl S-200 (70). Bovine factor Xa was isolated by gel filtration on Sephadex G-100 from the activation mixture of factor X by RW-XCP (77). The activation of protein C was conducted by the the same method as that of Ohno et al. (72). Measurement of Protein Concentration For purified preparations, protein concentrations were determined by absorbance measurement, employing,4^=15.5 for prothrombin (13); 12.4 for factor X (70; 14.9, for factor LX (75); 14.3 for factor IXa/3 (75); 13.7 for protein C (76); and 10.0 for proteins S (77). The concentration of protein Z was calculated by assuming an Agfa of 10. Measurement of Activity of Clotting Factors The activity of the activated protein C or factor Xa was measured as reported previously by using Boc-Leu-Ser-Thr-Arg-MCA or Boc-Ile-Glu-Gly- Arg-MCA, respectively, as a substrate (72, 18). A one-stage assay based on the partial thromboplastin time was used for the measurement of factor IX (19). One unit of factor IX activity was defined as the amount found in 1 ml of normal bovine plasma. The clotting activity of factor IXa^ was measured by the method of Fujikawa et al. (IS). Prothrombin was assayed by a twostage method (20). SDS-Polyacrylamide Gel Electrophoresis Electrophoresis on 8 % gel in the presence of SDS was performed by the method of Weber and Osbora (27). The gels were stained with Coomassie brilliant blue R-250. Concentration and Storage of Protein Large volumes of protein solutions (up to 1 liter) were concentrated by using Amicon ultrafiltration cells with PM 10 membranes and stored at -80 C. /. Bwchem.

3 PURIFICATION OF SIX VITAMIN K-DEPENDENT COAGULATION FACTORS 1349 Ammo Acid and NH t -Terminal Sequence Analyses Samples were hydrolyzed in 5.7 M HO in evacuated, sealed Pyrex tubes at 110 C for 24, 48, and 72 h. The analyses were performed with a Hitachi amino acid analyzer, model , by the method of Spackman (22). Tryptophan content was determined in hydrolyzates with 4 M methanesulfonic acid at 110 C for 24 h {23). y-carboxyglutamic acid content of protein S and protein Z was determined by the method of Fernlund et al. (24), using hydrolyzates of 2 to 3 nmol of protein in 100 //I of 3.75 M NaOH for 24 h at 110 C. The hydrolysate was neutralized by adding 100/d of 4 M HC1, lyophilized, dissolved in 0.2 M sodium citrate, ph 1.0, and analyzed on an amino acid analyzer. The NHj-terminal amino acid was determined by the manual Edman method, as described in detail previously (25). Purification Procedure General Approach and Methodology The purification scheme for six vitamin K-dependent proteins is described in two sections according to the flow sheet shown in Fig. 1. All procedures were performed at 4 C unless otherwise indicated. The conductivity of buffers and solutions was measured at 0 C. The data presented refer to the preparation of proteins from 15 liters of plasma. Preparation of Vitamin K-Dependent Protein Concentrate from Bovine Plasma Fresh bovine blood was collected at the slaughterhouse in polyethylene bottles and mixed with one-tenth volume of 0.11 M sodium citrate containing benzamidine- Sup Sup HC1 (1 g per liter). The blood was transported to the laboratory within an hour and centrifuged at 4,000 rpm for 20 min, using a Sorvall RC-3 centrifuge. Barium citrate adsorption was performed by a procedure similar to that of Moore et al. (26). One-tenth volume of 1.0 M barium chloride was added slowly to the plasma. The suspension was stirred gently for 60 min and centrifuged at 4,000 rpm for 10 min. The precipitate was washed with 0.1 M NaCl containing 0.01 M barium chloride, 1 mm benzamidine-hcl and 0.02 % NaN 3, and centrifuged again. The washing procedure was repeated twice. The washed barium citrate precipitate was suspended in I liter of 40% saturated ammonium sulfate solution. DFP was added to the solution to give a final concentration of 1 mm. The suspension was stirred at 4 C overnight. The precipitate was removed by centrifugation at 5,000 rpm for 30 min, and the supernatant was fractionated by adding saturated ammonium sulfate solution to give 67% saturation. The precipitate was collected by centrifugation at 5,000 rpm for 30 min in a Sorvall RC-3 centrifuge. The sediment was dissolved in ml of 0.05 M sodium phosphate buffer, ph 6.0, containing 0.2 M NaCl, 1 mm DFP, and 1 mm benzamidine-hcl, and dialyzed overnight against 20 liters of 0.05 M sodium phosphate buffer, ph 6.0, containing 0.2 M NaCl and 1 mm benzamidine-hcl. Insoluble material produced during dialysis was removed by centrifugation, and the clear supernatant solution was used as vitamin K-dependent protein concentrate. Cltrated plasma I Barium citrate precipitate Washing t elutlon with 40 * (NH <J 2SO 4 Sup I 67 * Fraction A Fraction B Blue- IHeparln- Sepnarose sephorose Ppt Ppt, saturation DEAE-Sephadex A-50 Fraction C IBUie- Sepnarose Protein S F. IX PT PT F. IX Protein C PT l Fraction D IBlue- I Septwros* F. X Protein Z Fig. 1. A systematic scheme for purification of bovine vitamin K-dependent proteins. The detailed procedures are described in the text. Sup, supernatant; Ppt, precipitate; F. IX, factor IX; PT, prothrombin; F. X, factor X. Vol. 97, No. 5, 1985

4 1350 N. HASHIMOTO, T MORITA, and S DEAE-Sephadex A-50 Chromatography The vitamin K-dependent protein concentrate (total absorbance at 280 nm = 4,760) obtained above was applied to a column (3.5 x 22 cm) of DEAESephadex A-50, equilibrated with 0.05 M sodium phosphate buffer, ph 6.0, containing 0.2 M NaCl and 1 mm benzamidine-hcl. After washing of the column with 1.5 liters of the equilibration buffer, linear salt gradient elution was performed with 1 liter each of the equilibration buffer and the same buffer containing 0.6 M NaCl. The flow rate was 90 ml per h and 16 ml fractions were collected. The elution profile is shown in Fig. 2. These fractions were pooled as indicated by solid bars and samples taken from the pooled fractions A, B, C, and D were subjected to SDS-PAGE. Fraction A contained the major portion of factor JX and protein S besides prothrombin. Fraction B contained mainly electrophoretically pure prothrombin, although factor IX activity was detectable in the same fraction. Fraction C contained mainly protein C, in addition to prothrombin. As shown in the inset to Fig. 2, two protein bands, which appeared to be a heavy chain and a light chain derived from protein C, were detected on reduced SDS-PAGE. The existence of protein C in fraction C was also confirmed by measuring the amidase activity of activated protein C towards Boc-Leu-Ser-Thr-Arg-MCA, after treatment with a-thrombin (12). On this column chromatography, factor X and protein Z were eluted in fraction D. Since protein Z does not show any enzyme activity, its detection was based on the electrophoretic behavior on reduced SDS-PAGE. As shown by line D in Fig. 2, a single protein band, which did not show any mobility change on unreduced and reduced SDS-PAGE, was detected, and this band was presumed to be protein Z. On reduced SDSPAGE (line D in Fig. 2), factor X yielded two protein bands as expected, which seemed to correspond to the heavy and light chains. B C D Isolation of Protein S and Factor IX from the Pooled Fraction A The pooled fraction A (Total /<280=744) obtained from the DEAE-Sephadex A-50 column was dialyzed overnight against 0.05 M Tris-HCl buffer, ph 7.4, containing 0. 1 M NaCl and 1 mm benzamidine, and applied to a column (2.8 x 25 cm) of Blue-Sepharose CL-6B, equilibrated with the buffer used for dialysis. The column was washed with the same buffer until PT 8 E 8 30 g E > o c o o < J o <-> s ~ 125 Fr No (I6ml/tub«) 150 Fig. 2. DEAE-Sephadex A-50 column chromatography of vitamin K-dependent protein concentrate. Details of the procedure and described in the text. The fractions A, B, C, and D indicated by solid bars were collected. The SDS-PAGE patterns of these pooled fractions and prothrombin (PT) used as a reference are shown in the insert. J. Biochem. A 15 IWANAGA

5 PURIFICATION OF SIX VITAMIN K-DEPENDENT COAGULATION FACTORS 1331 the absorbance at 280 nm of the breakthrough fraction (fraction a) reached a plateau (less than 0.1), then a small amount of factor IX, in addition to prothrombin, was eluted into fractions b 4 and c by 0.05 M Tris-HCl buffer, ph 7.4, containing 1 mm benzamidine and 4 M NaCI (Fig. 3A). The arrow in Fig. 3A indicates a change to elution buffer containing 4 M NaCI. This chromatographic procedure was essentially the same as that briefly reported for the preparation of protein S by Stenflo and Jonsson (27). However, they have not reported the chromatographic patterns in detail. On this column, protein S together with factor IX was eluted in the breakthrough fraction (fraction a) by the equilibration buffer, as shown in Fig. 3A. This procedure was repeated once to further purify the protein S and to separate factor IX (Fig. 3B). The pooled fraction a (Total A tso =\38) was applied to an identical column as shown in Fig. 3A. After the column had been washed with 0.05 M * The isolation of factor IX from fraction b was not performed, as its amount was so small m - O b C PT a *b i» \ e Fr. No. (8ml/tube) A 100 Tris-HCl, I mm benzamidine, ph 7.4, until the eluate showed an absorbance of less than 0.1 at 280 nm, factor IX was eluted by 0.05 M Tris-HCl buffer, ph 7.4, containing 1 mm benzamidine and 4 M NaCI at the position indicated by an arrow in Fig. 3B. Through these procedures, electrophoretically pure protein S, factor IX, and prothrombin were isolated in fractions d, e, and c, respectively. The fractions indicated by solid bars in Fig. 3B were pooled and concentrated. Isolation of Factor IX and Prothrombin from the Pooled Fraction B Factor IX and prothrombin were isolated from the pooled fraction B by using heparin-sepharose chromatography according to the method of Fujikawa et al. (19). The conditions and elution buffer used for the purification were identical with those described previously (19). Through these procedures, electrophoretically pure factor IX and prothrombin were recovered in good yield and in a functionally pure state. Isolation of Protein C from the Pooled Fraction C It is known that DEAE-Sephadex A-50 chromatography of the vitamin K-dependent protein 10 Q5 -- i -> 1 i rd i, Fr. No. (8 ml/tube) Fig. 3. Isolation of protein S from the pooled fraction A on a Blue-Sepharose CL-6B column. Details of the procedure are described in the text. A: The chromatogram for the pooled fraction A. B: The rechromatogram for the breakthrough fraction (fraction a). The fractions a, b, c, d, and e indicated by solid bars were collected. The SDS-PAGE patterns of these fractions are shown in the insert. e B 100 Vol. 97, No. 5, 1985

6 1352 N. HASHIMOTO, T. MORITA, and S. IWANAGA II Fr. No. (78ml/tube) Fig. 4. Isolation of protein C from the pooled fraction C on a Blue- Sepharose CL-6B column. Details of the procedure are described in the text. The fractions indicated by a solid bar were collected. The SDS- PAGE patterns of samples taken from fraction Nos. 30 (gel 1) and 120 (gel 2) are shown in the insert. concentrate can resolve protein C and prothrombin. However, the resolution is not very reproducible. We succeeded here in establishing a procedure for the isolation of protein C from the pooled fraction C, using a Blue-Sepharose CL-6B column. The pooled fraction C (Total /4 18O = 116) was first dialyzed overnight against 0.05 M imidazole-hcl buffer, ph 6.3, containing I ITIM benzamidine, and then applied to a column (2.8 x 25 cm) of Blue-Sepharose CL-6B, equilibrated with the same imidazole buffer. After washing of the column with 300 ml of the equilibration buffer, proteins were eluted with a linear gradient formed from 250 ml each of the same buffer and buffer containing 1 M NaCl, and 7.8 ml fractions were collected at a flow rate of 115 ml per h. The elution profile is shown in Fig. 4. The bulk of protein C was eluted in the breakthrough fraction and separated completely from prothrombin, as judged from the SDS-PAGE patterns of these components (shown in the inset to Fig. 4). Moreover, the protein C preparation thus isolated did not show any activity of factor IX or prothrombin, when the concentration of 0.1 mg per ml was used for the assay. Separation of Factor X and Protein Z from the Pooled Fraction D As described previously, the protein Z-containing fractions from the DEAE- Q4 02 O a 1 \ - a I/ - b i IM MOCJ / Fr. No. ( &5ml/tube) 20: Fig. 5. Separation of factor X and protein Z from the pooled fraction D on a Blue-Sepharose CL-6B column. Details of the procedure are described in the text. The fractions indicated by solid bars were collected. The SDS-PAGE patterns of these fractions are shown in the insert. Sephadex A-50 column overlapped with factor X. For separation of these components, a Blue-Sepharose CL-6B column was very effectively, as shown in Fig. 5. The pooled fraction D (total A 130 =! s o J. Biochem.

7 PURIFICATION OF SIX VITAMIN K-DEPENDENT COAGULATION FACTORS 1353 TABLE I. Summary of yield of vitamin K-dependent proteins from 15 liters of bovine plasma. values obtained from 3 experiments are shown. Component Fraction A Fraction B Fraction C Fraction D Yield (range) (mg) Protein S Factor IX ,300 1,600 Prothrombin Protein C Protein Z ) was dialyzed overnight against 0.05 M imidazole-hci buffer, ph 6.3, containing 1 rrim benzamidine, and applied to the column (1.6 x 16 cm) equilibrated with the buffer used for dialysis. After washing of the column with 100 ml of the equilibration buffer containing 0.1 M NaCI, proteins were eluted with a linear gradient formed from 100 ml each of the equilibration buffers containing 0.1 M and 0.7 M NaCI, and 8.5 ml fractions were collected at a flow rate of 72 ml per h. Through this procedure, the bulk of factor X was recovered in the breakthrough fraction, and protein Z, which showed a single band on both unreduced and reduced SDS-PAGE, was found in the adsorbed fraction. These fractions were pooled and concentrated. S IX PT C X Z Fig. 6. SDS-PAGE patterns of purified proteins. Approximately 10//g each of protein S (S), factor IX (IX), prothrombin (PT), protein C (C), factor X (X), and protein Z (Z) were subjected to SDS-PAGE. A summary of the overall yield (mg) of each component obtained from 15 liters of a single plasma pool is shown in Table I. The recovery of the various components isolated by the present method ular weights estimated under reducing conditions was reasonable as compared with those obtained were in good agreement with previously reported by reported methods for the individual purification values (/, 2). Protein S showed two bands, as of each component (3-5, 13, 14, 17, 19). All the reported by Stenflo and Jonsson (27). One of the steps mentioned above were repeated several times two bands is now known to be a hydrolysis produsing 15 to 20 liters of plasma and the results were uct, formed by limited proteolysis of the polypepfound to be reproducible. tide chain by a-thrombin or other plasma serine Homogeneity and Physicochemical Characteri- proteases during the purification procedure (28zation of the Purified Proteins The purified prep- 30). Our preparation of protein S cross-reacted arations of protein S, factor IX, prothrombin, with anti-bovine protein S antibody, when an protein C, factor X, and protein Z were subjected Ouchterlony test of the purified material was perto SDS-PAGE analysis. Under nonreducing conformed (data not shown). The specific activity of ditions, the isolated proteins except for protein S purified prothrombin was 1,200 NIH units per mg each gave a single band. Moreover, their molec- protein. The preparations of activated protein C Vol. 97, No. 5, 1985 Factor X RESULTS AND DISCUSSION The average

8 1354 N. HASHIMOTO, T MOR1TA, and S. JWANAGA and factor Xa had the same amidase activities as described in the literature (12, 18). Using the reported assay method (15), the preparation of factor IXa at 10ng/0.4ml of the assay mixture gave clotting times of s, compatible with that described by Fujikawa et al. (15). In Table II, the amino acid composition of the isolated protein Z is shown, in comparison with that recently reported by Petersen et al. (S). There were no major differences in amino acid composition between the two preparations. Moreover, the NH,-terminal tetrapeptide sequence, H- Ala-Gly-Ser-Tyr-, of the isolated protein Z was TABLE II Amino acid composition of protein Z. Amino acid Gla Asp Thr Ser Glu h Pro Gly Ala 1/2Cys Val Met lie Leu Tyr Phe Lys His Arg Trp Protein Z E 12.6" Residues per molecule N.D.i C s Protein Z>> 12.0' * Calculated from extrapolated or average values estimated on samples of 24, 48, and 72 h hydrolysates. Those for protein Z were calculated on the basis of 30 mol of aspartic acid/molecule. b Taken from the data of Petersen et al. (5). c 72 h hydrolysis value d Not determined. Calculated relative to aspartic acid by analysis of the alkaline hydrolyzate f Calculated relative to leucine (5). s Determined by the methanesulfonic acid method. h The glutamic acid content includes glutamine and y-carboxyglutamic acid. the same as that reported in the literature (5). The amino acid compositions of other components isolated here were also determined (data not shown) and the compositions were in good agreement with those of prothrombin (13), factor IX (19), factor X (14), protein S (IT), and protein C (3) described in the literature. In this work, we have extensively employed Blue-Sepharose chromatography. There are two reports that describe the purification of human factor X on blue-dextran agarose (31) and the interaction of prothrombin, factor IX, and factor VII with dextran-blue (32). The Blue-Sepharose column used to purify protein S from the pooled fraction A was similar to that described in the literature (27). We found here that this column was also very useful for the separation of protein C from the prothrombin-containing fraction and protein Z from the factor X-containing fraction. The isolation of these components from the DEAE- Sephadex A-50 pool was accomplished with only one additional step, except in the case of protein S. Thus, the method for the preparation of protein C and protein Z has been greatly improved in comparison with the previous method, in which a long column of DEAE-Sepharose and a rechromatography step were required for the isolation of these components from the DEAE-Sephadex A-50 pool (3, 5). The availability of this protocol for purification should facilitate further studies on the functional role of these proteins in the coagulation cascade. We wish to express our thanks to Dr. J Stenflo, University of Lund, Malmo, Sweden, for providing y- carboxyglutamic acid and identifying our protein S preparation immunologically. The assistance of Mrs. Sono Ezaki-Hashimoto in amino acid analysis is much appreciated. We also thank Miss Mizumo Akiyoshi for her expert secretarial assistance. REFERENCES 1. Davie, EW & Fujikawa, K. (1975) AMU. Rev. Biochem. 44, Jackson, CM. & Nemerson, Y. (1981) Annu Rev. Biochem. 49, Stenflo, J. (1976) J Biol. Chem. 251, DiScipio, R G, Hermodson. M.A., Yates, S.G., & Davie, E.W. (1977) Biochemistry 16, Peterson, T E, Thogersen, H.C., Sottrup-Jensen, L., Magnusson, S., & Jbrnvall, H. (1980) FEBS Lett. 114, J. Biochem.

9 PURIFICATION OF SIX VITAMIN K-DEPENDENT COAGULATION FACTORS March, S.C., Parikh, I., & Cuatrecasas, P. (1974) Anal. Biochem. 60, Holmberg, L., Bladh, B., & Astedt, B. (1976) Biochem. Biophys. Ada 445, Morita, T., Nishibe, H., Iwanaga, S., & Suzuki, T. (1974) /. Biochem. 76, Kisiel, W., Hermodson, M.A., & Davie, E.W. (1976) Biochemistry 15, 4901^ Amphlett, G.W., Byrne, R., & Castellino, F.J. (1979) /. Biol. Chem. 254, Morita, T. & Jackson, CM. (1980) in Vitamin K Metabolism and Vitamin K-Dependent Proteins (Suttie, J.W., ed) pp , University Park Press, Baltimore 12 Ohno, Y., Kato, H., Monta, T., Iwanaga, S., Takada, K., Sakakibara, S., & Stenflo, J. (1981) /. Biochem. 90, Owen, W.G., Esmon, C.T., & Jackson, C M. (1974) /. Biol. Chem. 249, Jackson, CM. & Hanahan, D. (1968) Biochemistry 7, 4506^ Fujikawa, K., Legaz, M.E., Kato, H., & Davie, E. (1974) Biochemistry 13, 4508-^ Kisiel, W., Ericsson, L.H., & Davie, E.W. (1976) Biochemistry 15, DiScipio, R.G. & Davie, E.W. (1979) Biochemistry 18, Monta, T., Kato, H., Iwanaga, S., Takada, K., Kimura, T., & Sakakibara, S. (1977) /. Biochem. 82, Fujikawa, K., Thompson, A.R., Legaz, M.E., Meyer, R G., & Davie, E.W. (1973) Biochemistry 12, Magnusson, S. (1970) Methods Enzymol. 19, Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, 4406^ Spackman, D.H., Stein, W.H., & Moore, S. (1958) Anal. Chem. 30, Simpson, R.J., Neuberger, M., & Liu, T.-Y. (1976) J. Biol. Chem. 251, Fernlund, P., Stenflo, J., Roepstorff, P., & Thompsen, J. (1975) /. Biol. Chem. 250, Edman, P. (1970) in Protein Sequence Determination (Needleman, S.B., ed.) pp , Springer-Verlag, Berlin 26. Moore, H.C., Lux, S.E., Malhotra, O.P., Bakerman, S., & Carter, J.R. (1965) Biochim. Biophys. Ada 111, Stenflo, J. & Jonsson, M. (1979) FEBS Lett. 101, Morita, T. & Iwanaga, S. (1982) Metabolism Disease (in Japanese) 19, Dahlback, B. (1983) Biochem. J. 209, Morita, T., Mizuguchi, J., & Iwanaga, S. /. Biochem. submitted 31. Vician, L. & Tishkoff, G H (1976) Biochim. Biophys. Ada 434, Schwart, A.C. & Hemker, H.C. (1970) Biochim. Biophys. Ada 222, Vol. 97, No. 5, 1985