THHOYBOSIS ;IESEARCH Printed in the United
vol. States
7, pp. 451-459, Pergamon Press,
1975 Inc.
PURIFICATION and CHARACTERIZATION of HtWAN FACTOR IX L-O. Andersson, H. Borg and M. Miller-Andersson AV KABI, Research Department, Biochemistry, Stockholm Sweden
(Received
10.6.1975; Accepted
in revised form 22.7.1975. by Editor A. Rimon)
ABSTRACT Human Factor IX has been purified from plasma by a procedure involving DEAE-Sephadex chromatography, affinity chromatography on heparin-Sepharose gel and gel filtration on Sephadex G-200. The final preparation was homogeneous according to a number of criteria and the specific activity corresponded to a 12 000 fold puri fication from plasma. The molecular weight was 72000 and only tyru sine was found as the N-terminal amino acid. The amino acid composition resembled that of prothrombin. The carbohydrate content was 22.8 %. No change in molecular weight was observed upon polyacrylamide gel electrophoresis in 8 M urea containing mercaptoethanol indicating that Factor IX is a single polypeptide chain crosslinked by disulphide bonds.
INTRODUCTION Many attempts have been made to isolate Factor IX. The separation problems provide considerable difficulties however, since the plasma concentration is less than 10 ug(ml and the physicochemical properties of the vitamin K dependent coagulation factors, II, VII, IX and X are very similar. Various Factor IX concentrates fwe been available for a considerable time but although sufficiently concentrated for clinical use they are relatively crude preparations. The content of Factor IX in these concentrates is about l-2 % of the total protein content and considerable amounts of Factors II and X are present. Tn this study a procedure has been developed where Factor IX is isolated in pure state. Affinity chromatography on heparin-Sepharose gel is the main
451
452
HUMAN FACTOR
IX F'URIFICATION
separation step used. A preliminary report of this investigation has been given (LL
Lyophilized lieparinwas obtained from AB Vitrum, Stockholm. Sepharose 4B gel and DEAE-Sephadex A-50 were products from Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was purchased from Fluka, Switzerland. The antisera used were obtained from Behringwerke, Hoechst, West Germany. All other chemicals used were the purest commercially available, usually reagent grade. Specific reagent for prothrombin assay (Ampoule P) was obtained from AB Imco Stockholm, Sweden. Factor X Deficient Substrate Plasma was obtained from Merz and Dade AG, Berne Switzerland (human congenital deficient plasma). Stypven Russell Viper Venom was obtained from Wellcome Reagents Ltd, Beckenham, England. Bell and Alton platelet substitute was obtained from Diagnostic Reagents Ltd, Thame Oxon, England. Blood plasma. Pooled ml of cltrated blood 30 minutes at 2000 g and dispensed into 5 -7oOc.
normal plasma was prepared in the following way: Thirty from 12 healthy persons were taken and centrifuged for and the plasma-removed. The plasma samples were-pooled ml plastic tubes which were quick-frozen and kept at
F IX deficient plasma was obtained from persons with severe hemophilia B and to whom no plasma or Factor IX concentrate had been given during the past two months. Preparation of heparin-Sepharose gel Twenty g of cyanogen bromide was dissolved in 500 ml of a 0.4 % heparin solution in water. Then, 500 ml of washed Sepharose 4B gel was mixed with the solution. Subsequent reactions were performed in an ice bath, the pH was raised to 11.0 and maintained there by the dropwise addition of 5 M NaOH, with continous stirring for about seven minutes. The pH then gradually decreased to about 8 and the mixture was stirred overnight at room temperature. The gel was then treated with 250 ml of 0.1 M ethanolamine solution to block any remaining reactive groups. Finally, the gel was washed extensively with distilled water, 0.1 M sodium acetate buffer pH 4.7 and 0.5 M NaHC03 solution. The amount of heparin coupled was determined by amino acid analysis for glu-cosamine after hydrolysis in 2 M HCl at llO°C for 24 hours. The gels used contained between 0.7 - 1.1 mg of heparin per ml of gel. Assays of clotting factors Factor IX activity was assayed itia one stage method as described by Veltkamp (2). An incubation mixture was prepared,containing platelet poor Factor IX deficient plasma, kaolin, brain thromboplastin and a diluted sample of norma plasma or the fraction to be tested. After 18 minutes incubation at 37'C, Ca + was added and the clotting time registered. For preparation of a standard
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HIJMAX FACTOR
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PUXIFICATION
453
curve a series of dilutions of normal plasma was tested. One unit Factor IX was taken as tb_eFactor IX content in 1 ml of normal plasma. Prothrombin was assayed in a two-stage system based on an activated mixture of all the intrinsic coagulation factors except prothrombin. Commercial reagents obtained from AB Imco were used and the assay was performed as described by Noren (3). Factor X was assayed using Russel Viper Venom as described by Denson (4). Polyacrylamide gel electrophoresis Disc polyacrylamide gel electrophoresis was carried out at pH 8.5 essentially as described by Davies (5). The polymerization was performed in 0.05 M tris (hydroxymethyl) aminomethane, 0.05 M glycine buffer pH 8.5. Ammonium persulphate (0.2 % w/v) and dimethylaminopropionitrile (0.02 % w/v) were used as catalysts. After electrophoresis the gels were stained with amido black and were destained by shaking in 10 % acetic acid. The Gradipore polyacrylamide gel electrophoresis runs were performed as described by Andersson et al. (6) in 0.05 M Tris buffer pH 8.5 at 90 Volt and 6'C for 18 hours. In the molecular weight determinations a series of plasma proteins was used to obtain the linear log Mw-migration distance calibration curve. Immunoelectrophoresis Immunoelectrophoresis on agarose gels was performed essentially as described by Scheidegger et al. (7). Amino acid analysis Hydrolysis was performed for 24 h at llO°C in thoroughly evacuated glasstubes using 6 N HCl. The hydrolysate was analyzed with a Durrum D-500 analyzer. For determination of the cystine content performic acid oxidation was used. End group determination Amino terminal determination was performed manually by the direct pheny 1 isothiocyanate Edman method as described by Fryklund et al (8). Carbohydrate determination The content of neutral sugar was determined by the phenol-H,SO, method ( 9). N-acetyl glucosamine and N-acetylgalactosamine contents werb obtained from the amino acid analysis using correction factors for destruction during hydrolysis. Sialic acid was determined using thio-barbituric acid according to Warren (10). RESULTS
Purification procedure: The starting material used was plasma after precipitation of Cohn fraction I by 8 % ethanol. To 6 litres of this was added 200 ml of swollen DEAR-Sephadex-A-50 gel and the mixture was stirred for half an hour at O'C. The gel was filtered and washed with 0.3 M NH4HC03 buffer pH 8.0 followed by elution of the Factor IX containing material with 0.75 M NH4HC03 buffer pH 8.0. The eluted material contained 1 unit of Factor IX
H-U-MANFACTOR
454
IX PURIF'ICMXON
Vo1.7,No.3
activity per mg of prote,in.Following lyophilization an almost saltfree preparation was obtained. This was dissolved in CL.05M Tris, 0.02 M citrate, 0,l M NaCl .buEfer pH7.4 yielding a protein solution of 15 mg[ml. The solution was applied to a column of beparin-Sepharose gel equilibrated with the same buffer. After elution with one column volume of buffer desorption was achieved using NaCl gradient (0.15 M to 2 Ml in 0.05 M acetate, 0.02 M citrate buffer pH 5..Figure 1 shows the elution diagram obtained. Factor IX elutes in the later part of the main peak. A considerable degree of purification is obtained in this step since the specific activity is strongly increased as evident from Table 1. Most of the Factors II and X are also removed in this step. *ABLE Purification
Activity units/ml
4500
PLasma DEAF?Seph.d.X batch-fracrionation
230
1:s~ Hepacin-Sepharose gel affinity chromate-
L of
Facror
IX
Purificarian
Specific accivi:y unir5/A2So
0.93
4200
I
LZ.9
0.78
2910
250
8.2
6.9
2030
2:nd Heparin-Sqharore g.e* affinity ChrwxaLo-
110
8.L
11.5
,100
L 200
Septmdex
0 200
160
6.1
87
,070
1500
‘EC-Sephadex :o,umn procedure
58
16.0
70
600
CaPhY
gelfilrrarioo
1‘2
930
12200
*2801
1
3
NaCl
2 .2
20-
Fncy 1
.l
E . (I) .Z
10 5 s .e > .ti 0.5
1.0 Elution-- volume,
1.5
liter
FIG. 1 Elution diagram of heparin-Sepharose affinity chromatography of Factor IX concentrate. Desorption was performed by linear salt gradient elution from 0.15 NaCl in 0.05 M acetate, 0.02 M citrate buffer pH 5.0. The dimensions of the column were 5 x 27 cm.
HUMAN
FACTOR
M
PURIFICATION
455
Figure 2 shows the gradipore polyacrylamide gel electrophoresis pattern of material from theyarious separation steps. Since there is some concentration dependent nonspecific adsorption to the heparin-Sepliarosegel, the. third separation step is a rerun which removes the last traces of Factors IT and X. A further increase in specific activity can be seen in Table 1. After concentration of the material by ammonium sulphate precipitation at 55 % saturation and pH 5.0 a 2 % solution of protein is applied to a Sephadex G-200 column. The gel filtration elution diagram obtained is shown in Figure 3. The Factor IX activity peak coincides witli the final W-absorbing peak at an elution volume corresponding to a molecular weight of 82 000, when compared with a number of plasma proteins of known molecular weights.
1.0 A200
0.5
0.6
0.8
1.0
Elution volume, liter Fig.2. Gradipore polyacrylamide gel electrophoresis patterns of Factor IX containing material in various stages of purification. 1. Plasma. 2. Eluate from DEAE-Sephadex fractionation batch procedure. 3. Eluate after first heparin -Sepharose step. 4. Eluate after G 200 gel filtration step. 5. Pure Factor IX. Fig.3. Sephadex G 200 gel filtration elution diagram of Factor IX derived from heparin-Sepharose fractionation. The buffer used was 0.05 M Tris, 0.01 M citrate, 0.10 M NaCl of pH 7.4.
456
HUMAN
FACTOR
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PURIFICATION
Vo1.7,No.3
The final separation step is an ion exchange chromatography on DEAE-Sephadex A 50 in 0.05 M Tris 0.02 M citrate buffer pH7.9 using salt gradient elution from 0.05 M NaCl to 0.55 M NaCl which removes all the remaining contaminants. The gradipore polyacrylamide gel electrophoresis shows only one band as evident from Figure 2. Polyacrylamide gel electrophoresis in 7 % gel also revealed one band. Inrmunoelectrophoresisagainst anti-whole serum did not show any precipitin line even at higK sample concentrations. This is not surpris:. ing as the antiserum can be expected to Ke devoid of antibodies towards Factor IX due to the low concentration in plasma. No Factor II or Factor X activity could be detected. One of the main problems- in the purification was to avoid activation. Our first series of purification experiments resulted often in activation of Factor IX, observed as Factor IX active material behind the Factor IX peak in the Sephadex G 200 gel filtration. We found out later that it was essential t'operform all the separation procedures at a temperature below +5'C and as fast as possible so that no solutions were allowed to stand. Platelet free or platelet poor plasma was found to be the best starting material. Taking this into account it was possible to avoid activation. To confirm that our purified Factor IX behaved as the Factor IX in plasma two gelfiltration experiments on a 100 cm long Sephadex G 200 column were performed where plasma and the pure Factor IX preparation were run. Activity determinations showed that the peak of the activity eluted at exactly the same volume, slightly before albumin, in both cases. Chemical characterization
of Factor IX
The molecular weight estimated from the gel filtration runs was 82 000. However it is well known that gel filtration in ordinary buffer solutions can give erroneous results in certain cases depending on conformational effects. To study this further, molecular weight determination was performed by Gradipore polyacrylamide gel electrophoresis (6). Two 18 hour runs were performed using in one instance d.05 M Tris-HCl buffer pH 8.5 and in the other 0.05 M Tris - HCl buffer pH 8.5 containing 8 M urea. The run in Tris buffer alone yielded a molecular weight of 71 000 and the corresponding run in Tris buffer-urea 72 000. The Factor IX band appeared close to albumin. Determination of the number of peptide chains was performed in the presence of mercaptoethanol. Gradipore polyacrylamide gel electrophoresis in 0.05 M Tris-HCl buffer pH 8.5 containing 8 M urea and 0.1 M mercaptoethanol yielded a value of 68 000 for themolecular weight. In polyacrylamide gel electrophoresis in 0.05 M Tris-HCl buffer pH 8.5 containing 1 % SDS and 0.1 M mercaptoethanol the mobility of the Factor IX corresponded to a molecular weight of 72:OOO when compared to a number of other plasma proteins as references. Those runs were performed in 7 % polyacrylamide gel. When comparing the different values obtained for the molecular weight it appears likely that the gel filtration value is somewhat high and that the true value probably is close to 72 000. However it is possible that the value is even lower because proteins with high carbohydrate contents often give high values for molecular weight in polyacrylamid-electrophoresis.
JXJMAX FACTOR
M
457
F'URIFICXION
TABLE 2. Amino acid composition
Amino acid
Residues per 100 residues
Estimated number of residues per ?fw 55 000
Lysine
6.13
Histidine
2.28
11
4.15
21
11.10
55
Threonine
6.70
34
Serine
6.42
32
13.09
65
Proline
4.57
23
Glycine
8.52
43
Alanine
5.48
27
Half-cystine
5.20
26
Valine
7.48
37
Methionine
0.89
4
Isoleucine
4.44
22
Leucine
5.73
29
Tyrosine
3.49
18
Phenylalanine
4.33
22
Arginine Aspartic acid
Glutamic acid
31
t Determination of the content of neutral sugar of pure Factor IX yielded the value 6.5 %. The content of N-acetyl-glucosamine was 6.8 % and no N-acetylgalactosamine was found. The content of sialic acid was 9.5 %. The amino acid composition data are shown in Table 2. The values given are the averages of two separate determinations on each of two Factor IX preparations. The extinction coefficient was determined by dissolving a known amount of dry and saltfree Factor IX in 0.1 M Tris-HCl buffer pH 7.4 and measuring the 1% absorption. The value obtained was E = 10.7. 28Onm Since the tyrosine content is known from amino acid analysis the presence of about 10 tryptophan residues in Factor IX is indicated, Amino-terminal determination on pure Factor IX yielded tyrosine as the only N-terminal amino acid. In the earlier stages of the work when activation often occured glycine and occasionally lysine were found as additional N-terminals. In the final procedure none of these could be detected.
458
HUMAN
FACTOR
IX PURIFICATION
DISCUSSLON The two main problems inyolved in the purification of the vitamin K-dependent coagulation factors- are to separate them from each other and to avoid activation. In the present method foF the purification of Factor IX the most important step is the heparin-Sepharose affinity chromatography where Factor IX is separated from prothrombin and Factor X. Heparin-agarose chromatography has also been used in the purification of bovine Factor IX (11) but the separation was performed in the presence of Ca2+ to achieve complete separation from Factor II. This was not necessary with human Factor IX. A possible explanation is that the elution is performed at a lower pH than was the case with the bovine protein. However, to achieve complete removal of prothrombin the procedure had to be repeated. The activation problems were solved mainly by performing all steps in the cold and working as rapidly as possible. The low pH used in the heparin-Sepharose affinity chromatography step and the absence of Ca2+ in any step are probably also important for avoiding activation. The binding of Factor IX to heparin appears to be a highly specific reaction. It occurs over a broad pH-range including both neutral and slightly alkaline pH where both heparin and Factor IX are negatively changed and should repel each other if only electrostatic forces were involved. Preliminary experiments showed no binding of Factor IX to dextran sulphate Sepharose or SPSephadex at neutral pH further supporting the concept of specific binding of heparin to Factor IX. It is likely that both the charged sulphate groups and the carbohydrate structures of heparin are involved in the binding. The chemical characterization of pure Factor IX shows that it is a glycoprotein with a molecular weight of about 72 000 composed of a single peptide chain crosslinked by 13 disulphide bridges. The amino acid composition is that of normal globular protein but the fairly large proportion of hydrophilic amino acids should be noted. The amino acid composition is similar to that of human prothrombin (12) but some notable differences are found, especially the higher contents of cystine and lysine and the lower content of tyrosine in Factor IX as compared to prothrombin. The aminoterminal amino acids are also different being tyrosine for Factor IX and alanine for prothrombin. Comparison of bovine (11) and human Factor IX show that the amino acid compositions are very similar and there is also good agreement with respect to the carbohydrate content. The aminoterminal amino acid is also the same. There is however a considerable difference in molecular weight, 55 000 for the bovine factor and 72 000 for the human. Recently another method for purification of human Factor IX was published (13). The pure protein obtained showed about the same molecular weight as the material obtained in this study but there are important differences in other respects. The amino-terminal acids are different being tyrosine in this study and glycine in the other study. There are also significant differences in the amino and carbohydrate compositions. ACKNOWLEDGMENT Thanks are due to Dr Linda Fryklund for valuable discussions and for performing the aminoterminal analyses. Thanks are also due to Mrs Anita Linder for excellent technical assistance and to Mrs Karin Brundin for typing the manuscript.
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Ix PURIFICATION
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REFERENCES 1.
ANDERSSON,
L-O., BORG, H. and MILLER-ANDERSSON, M. Purification of Factor IX by Affinity Chromatography. Abstract from IVth International Congress on Thrombosis and HaemostasisT Wien, 1973, p 142.
2. VELTKAMP, J.J., DRION, E.F. and LOELIGER, E.A. Detection of the Carrier State in Hereditary Coagulation Disorders I. Thromb. Diath. Haemorrhag. 19-20, 279, 1968. 3. NOREN, I. Specific Assay of Prothrombin. Stand. J. Clin. Lab. Invest. 25, 47, 1970. 4. DENSON, K.W.E. The Specific Assay of Prower-Stuart Factor and Factor VII. Acta Haematologica 25, 105. 5. DAVIES, B.J. Disc. electrophoresis-II. Method and Application to Human Serum Proteins. Ann. N.Y. Acad. Sci.121, 404, 1964. 6. ANDERSSON, L-O., BORG, H. and MIKAELSSON, M. Molecular Weight Estimations of Protein by Electrophoresis in Polyacrylamide gels of Graded Porosity. FEBS Letters, 20, 199, 1972. 7. SCHEIDEGGER, J.J. Une Micromethode de L'immunoelectrophorese. Int Arch. Allergy. Appl. Irxaunol.7, 103, 1955. 8. FRYKLUND, L., EAKER, D. and KARLSSON, E. Amino Acid Sequences of the two Principal Neurotoxins of Enhydrina Scistosa Venom. Biochemistry, 11, 4633, 1972. 9. DUBOIS, M., GILLES, K.A., HAMILTON, J.K.. REBERS, P.A. and SMITH, F. Calorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 28, 350, 1956. 10. WARREN, L. The Thiobarbituric Acid Assay of Sialic Acids. J. Biol. Chem. 234, 1971, 1959. 11. FUJIKAWA, K., THOMPSON, A.R., LEGAZ, M.E., MEYER, R.G. and DAVIE, E.W. Isolation and Characterization of Bovine Factor IX. Biochemistry, 12, 4938, 1973. 12. KISIEL, W. and HANAHAN, D.J. Purification and Characterization of Human Factor II. Biochim, Biophys. Acta. 304, 103, 1973. 13. aSTERLiD,B. and FLENGSRUD, R. Purification and Some Characteristics of the Coagulation Factor IX from Human Plasma. Biochem. J. 145, 469, 1975.
vol. States
7, pp. 451-459, Pergamon Press,
1975 Inc.
PURIFICATION and CHARACTERIZATION of HtWAN FACTOR IX L-O. Andersson, H. Borg and M. Miller-Andersson AV KABI, Research Department, Biochemistry, Stockholm Sweden
(Received
10.6.1975; Accepted
in revised form 22.7.1975. by Editor A. Rimon)
ABSTRACT Human Factor IX has been purified from plasma by a procedure involving DEAE-Sephadex chromatography, affinity chromatography on heparin-Sepharose gel and gel filtration on Sephadex G-200. The final preparation was homogeneous according to a number of criteria and the specific activity corresponded to a 12 000 fold puri fication from plasma. The molecular weight was 72000 and only tyru sine was found as the N-terminal amino acid. The amino acid composition resembled that of prothrombin. The carbohydrate content was 22.8 %. No change in molecular weight was observed upon polyacrylamide gel electrophoresis in 8 M urea containing mercaptoethanol indicating that Factor IX is a single polypeptide chain crosslinked by disulphide bonds.
INTRODUCTION Many attempts have been made to isolate Factor IX. The separation problems provide considerable difficulties however, since the plasma concentration is less than 10 ug(ml and the physicochemical properties of the vitamin K dependent coagulation factors, II, VII, IX and X are very similar. Various Factor IX concentrates fwe been available for a considerable time but although sufficiently concentrated for clinical use they are relatively crude preparations. The content of Factor IX in these concentrates is about l-2 % of the total protein content and considerable amounts of Factors II and X are present. Tn this study a procedure has been developed where Factor IX is isolated in pure state. Affinity chromatography on heparin-Sepharose gel is the main
451
452
HUMAN FACTOR
IX F'URIFICATION
separation step used. A preliminary report of this investigation has been given (LL
Lyophilized lieparinwas obtained from AB Vitrum, Stockholm. Sepharose 4B gel and DEAE-Sephadex A-50 were products from Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was purchased from Fluka, Switzerland. The antisera used were obtained from Behringwerke, Hoechst, West Germany. All other chemicals used were the purest commercially available, usually reagent grade. Specific reagent for prothrombin assay (Ampoule P) was obtained from AB Imco Stockholm, Sweden. Factor X Deficient Substrate Plasma was obtained from Merz and Dade AG, Berne Switzerland (human congenital deficient plasma). Stypven Russell Viper Venom was obtained from Wellcome Reagents Ltd, Beckenham, England. Bell and Alton platelet substitute was obtained from Diagnostic Reagents Ltd, Thame Oxon, England. Blood plasma. Pooled ml of cltrated blood 30 minutes at 2000 g and dispensed into 5 -7oOc.
normal plasma was prepared in the following way: Thirty from 12 healthy persons were taken and centrifuged for and the plasma-removed. The plasma samples were-pooled ml plastic tubes which were quick-frozen and kept at
F IX deficient plasma was obtained from persons with severe hemophilia B and to whom no plasma or Factor IX concentrate had been given during the past two months. Preparation of heparin-Sepharose gel Twenty g of cyanogen bromide was dissolved in 500 ml of a 0.4 % heparin solution in water. Then, 500 ml of washed Sepharose 4B gel was mixed with the solution. Subsequent reactions were performed in an ice bath, the pH was raised to 11.0 and maintained there by the dropwise addition of 5 M NaOH, with continous stirring for about seven minutes. The pH then gradually decreased to about 8 and the mixture was stirred overnight at room temperature. The gel was then treated with 250 ml of 0.1 M ethanolamine solution to block any remaining reactive groups. Finally, the gel was washed extensively with distilled water, 0.1 M sodium acetate buffer pH 4.7 and 0.5 M NaHC03 solution. The amount of heparin coupled was determined by amino acid analysis for glu-cosamine after hydrolysis in 2 M HCl at llO°C for 24 hours. The gels used contained between 0.7 - 1.1 mg of heparin per ml of gel. Assays of clotting factors Factor IX activity was assayed itia one stage method as described by Veltkamp (2). An incubation mixture was prepared,containing platelet poor Factor IX deficient plasma, kaolin, brain thromboplastin and a diluted sample of norma plasma or the fraction to be tested. After 18 minutes incubation at 37'C, Ca + was added and the clotting time registered. For preparation of a standard
Vol.7
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HIJMAX FACTOR
IX
PUXIFICATION
453
curve a series of dilutions of normal plasma was tested. One unit Factor IX was taken as tb_eFactor IX content in 1 ml of normal plasma. Prothrombin was assayed in a two-stage system based on an activated mixture of all the intrinsic coagulation factors except prothrombin. Commercial reagents obtained from AB Imco were used and the assay was performed as described by Noren (3). Factor X was assayed using Russel Viper Venom as described by Denson (4). Polyacrylamide gel electrophoresis Disc polyacrylamide gel electrophoresis was carried out at pH 8.5 essentially as described by Davies (5). The polymerization was performed in 0.05 M tris (hydroxymethyl) aminomethane, 0.05 M glycine buffer pH 8.5. Ammonium persulphate (0.2 % w/v) and dimethylaminopropionitrile (0.02 % w/v) were used as catalysts. After electrophoresis the gels were stained with amido black and were destained by shaking in 10 % acetic acid. The Gradipore polyacrylamide gel electrophoresis runs were performed as described by Andersson et al. (6) in 0.05 M Tris buffer pH 8.5 at 90 Volt and 6'C for 18 hours. In the molecular weight determinations a series of plasma proteins was used to obtain the linear log Mw-migration distance calibration curve. Immunoelectrophoresis Immunoelectrophoresis on agarose gels was performed essentially as described by Scheidegger et al. (7). Amino acid analysis Hydrolysis was performed for 24 h at llO°C in thoroughly evacuated glasstubes using 6 N HCl. The hydrolysate was analyzed with a Durrum D-500 analyzer. For determination of the cystine content performic acid oxidation was used. End group determination Amino terminal determination was performed manually by the direct pheny 1 isothiocyanate Edman method as described by Fryklund et al (8). Carbohydrate determination The content of neutral sugar was determined by the phenol-H,SO, method ( 9). N-acetyl glucosamine and N-acetylgalactosamine contents werb obtained from the amino acid analysis using correction factors for destruction during hydrolysis. Sialic acid was determined using thio-barbituric acid according to Warren (10). RESULTS
Purification procedure: The starting material used was plasma after precipitation of Cohn fraction I by 8 % ethanol. To 6 litres of this was added 200 ml of swollen DEAR-Sephadex-A-50 gel and the mixture was stirred for half an hour at O'C. The gel was filtered and washed with 0.3 M NH4HC03 buffer pH 8.0 followed by elution of the Factor IX containing material with 0.75 M NH4HC03 buffer pH 8.0. The eluted material contained 1 unit of Factor IX
H-U-MANFACTOR
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IX PURIF'ICMXON
Vo1.7,No.3
activity per mg of prote,in.Following lyophilization an almost saltfree preparation was obtained. This was dissolved in CL.05M Tris, 0.02 M citrate, 0,l M NaCl .buEfer pH7.4 yielding a protein solution of 15 mg[ml. The solution was applied to a column of beparin-Sepharose gel equilibrated with the same buffer. After elution with one column volume of buffer desorption was achieved using NaCl gradient (0.15 M to 2 Ml in 0.05 M acetate, 0.02 M citrate buffer pH 5..Figure 1 shows the elution diagram obtained. Factor IX elutes in the later part of the main peak. A considerable degree of purification is obtained in this step since the specific activity is strongly increased as evident from Table 1. Most of the Factors II and X are also removed in this step. *ABLE Purification
Activity units/ml
4500
PLasma DEAF?Seph.d.X batch-fracrionation
230
1:s~ Hepacin-Sepharose gel affinity chromate-
L of
Facror
IX
Purificarian
Specific accivi:y unir5/A2So
0.93
4200
I
LZ.9
0.78
2910
250
8.2
6.9
2030
2:nd Heparin-Sqharore g.e* affinity ChrwxaLo-
110
8.L
11.5
,100
L 200
Septmdex
0 200
160
6.1
87
,070
1500
‘EC-Sephadex :o,umn procedure
58
16.0
70
600
CaPhY
gelfilrrarioo
1‘2
930
12200
*2801
1
3
NaCl
2 .2
20-
Fncy 1
.l
E . (I) .Z
10 5 s .e > .ti 0.5
1.0 Elution-- volume,
1.5
liter
FIG. 1 Elution diagram of heparin-Sepharose affinity chromatography of Factor IX concentrate. Desorption was performed by linear salt gradient elution from 0.15 NaCl in 0.05 M acetate, 0.02 M citrate buffer pH 5.0. The dimensions of the column were 5 x 27 cm.
HUMAN
FACTOR
M
PURIFICATION
455
Figure 2 shows the gradipore polyacrylamide gel electrophoresis pattern of material from theyarious separation steps. Since there is some concentration dependent nonspecific adsorption to the heparin-Sepliarosegel, the. third separation step is a rerun which removes the last traces of Factors IT and X. A further increase in specific activity can be seen in Table 1. After concentration of the material by ammonium sulphate precipitation at 55 % saturation and pH 5.0 a 2 % solution of protein is applied to a Sephadex G-200 column. The gel filtration elution diagram obtained is shown in Figure 3. The Factor IX activity peak coincides witli the final W-absorbing peak at an elution volume corresponding to a molecular weight of 82 000, when compared with a number of plasma proteins of known molecular weights.
1.0 A200
0.5
0.6
0.8
1.0
Elution volume, liter Fig.2. Gradipore polyacrylamide gel electrophoresis patterns of Factor IX containing material in various stages of purification. 1. Plasma. 2. Eluate from DEAE-Sephadex fractionation batch procedure. 3. Eluate after first heparin -Sepharose step. 4. Eluate after G 200 gel filtration step. 5. Pure Factor IX. Fig.3. Sephadex G 200 gel filtration elution diagram of Factor IX derived from heparin-Sepharose fractionation. The buffer used was 0.05 M Tris, 0.01 M citrate, 0.10 M NaCl of pH 7.4.
456
HUMAN
FACTOR
IX
PURIFICATION
Vo1.7,No.3
The final separation step is an ion exchange chromatography on DEAE-Sephadex A 50 in 0.05 M Tris 0.02 M citrate buffer pH7.9 using salt gradient elution from 0.05 M NaCl to 0.55 M NaCl which removes all the remaining contaminants. The gradipore polyacrylamide gel electrophoresis shows only one band as evident from Figure 2. Polyacrylamide gel electrophoresis in 7 % gel also revealed one band. Inrmunoelectrophoresisagainst anti-whole serum did not show any precipitin line even at higK sample concentrations. This is not surpris:. ing as the antiserum can be expected to Ke devoid of antibodies towards Factor IX due to the low concentration in plasma. No Factor II or Factor X activity could be detected. One of the main problems- in the purification was to avoid activation. Our first series of purification experiments resulted often in activation of Factor IX, observed as Factor IX active material behind the Factor IX peak in the Sephadex G 200 gel filtration. We found out later that it was essential t'operform all the separation procedures at a temperature below +5'C and as fast as possible so that no solutions were allowed to stand. Platelet free or platelet poor plasma was found to be the best starting material. Taking this into account it was possible to avoid activation. To confirm that our purified Factor IX behaved as the Factor IX in plasma two gelfiltration experiments on a 100 cm long Sephadex G 200 column were performed where plasma and the pure Factor IX preparation were run. Activity determinations showed that the peak of the activity eluted at exactly the same volume, slightly before albumin, in both cases. Chemical characterization
of Factor IX
The molecular weight estimated from the gel filtration runs was 82 000. However it is well known that gel filtration in ordinary buffer solutions can give erroneous results in certain cases depending on conformational effects. To study this further, molecular weight determination was performed by Gradipore polyacrylamide gel electrophoresis (6). Two 18 hour runs were performed using in one instance d.05 M Tris-HCl buffer pH 8.5 and in the other 0.05 M Tris - HCl buffer pH 8.5 containing 8 M urea. The run in Tris buffer alone yielded a molecular weight of 71 000 and the corresponding run in Tris buffer-urea 72 000. The Factor IX band appeared close to albumin. Determination of the number of peptide chains was performed in the presence of mercaptoethanol. Gradipore polyacrylamide gel electrophoresis in 0.05 M Tris-HCl buffer pH 8.5 containing 8 M urea and 0.1 M mercaptoethanol yielded a value of 68 000 for themolecular weight. In polyacrylamide gel electrophoresis in 0.05 M Tris-HCl buffer pH 8.5 containing 1 % SDS and 0.1 M mercaptoethanol the mobility of the Factor IX corresponded to a molecular weight of 72:OOO when compared to a number of other plasma proteins as references. Those runs were performed in 7 % polyacrylamide gel. When comparing the different values obtained for the molecular weight it appears likely that the gel filtration value is somewhat high and that the true value probably is close to 72 000. However it is possible that the value is even lower because proteins with high carbohydrate contents often give high values for molecular weight in polyacrylamid-electrophoresis.
JXJMAX FACTOR
M
457
F'URIFICXION
TABLE 2. Amino acid composition
Amino acid
Residues per 100 residues
Estimated number of residues per ?fw 55 000
Lysine
6.13
Histidine
2.28
11
4.15
21
11.10
55
Threonine
6.70
34
Serine
6.42
32
13.09
65
Proline
4.57
23
Glycine
8.52
43
Alanine
5.48
27
Half-cystine
5.20
26
Valine
7.48
37
Methionine
0.89
4
Isoleucine
4.44
22
Leucine
5.73
29
Tyrosine
3.49
18
Phenylalanine
4.33
22
Arginine Aspartic acid
Glutamic acid
31
t Determination of the content of neutral sugar of pure Factor IX yielded the value 6.5 %. The content of N-acetyl-glucosamine was 6.8 % and no N-acetylgalactosamine was found. The content of sialic acid was 9.5 %. The amino acid composition data are shown in Table 2. The values given are the averages of two separate determinations on each of two Factor IX preparations. The extinction coefficient was determined by dissolving a known amount of dry and saltfree Factor IX in 0.1 M Tris-HCl buffer pH 7.4 and measuring the 1% absorption. The value obtained was E = 10.7. 28Onm Since the tyrosine content is known from amino acid analysis the presence of about 10 tryptophan residues in Factor IX is indicated, Amino-terminal determination on pure Factor IX yielded tyrosine as the only N-terminal amino acid. In the earlier stages of the work when activation often occured glycine and occasionally lysine were found as additional N-terminals. In the final procedure none of these could be detected.
458
HUMAN
FACTOR
IX PURIFICATION
DISCUSSLON The two main problems inyolved in the purification of the vitamin K-dependent coagulation factors- are to separate them from each other and to avoid activation. In the present method foF the purification of Factor IX the most important step is the heparin-Sepharose affinity chromatography where Factor IX is separated from prothrombin and Factor X. Heparin-agarose chromatography has also been used in the purification of bovine Factor IX (11) but the separation was performed in the presence of Ca2+ to achieve complete separation from Factor II. This was not necessary with human Factor IX. A possible explanation is that the elution is performed at a lower pH than was the case with the bovine protein. However, to achieve complete removal of prothrombin the procedure had to be repeated. The activation problems were solved mainly by performing all steps in the cold and working as rapidly as possible. The low pH used in the heparin-Sepharose affinity chromatography step and the absence of Ca2+ in any step are probably also important for avoiding activation. The binding of Factor IX to heparin appears to be a highly specific reaction. It occurs over a broad pH-range including both neutral and slightly alkaline pH where both heparin and Factor IX are negatively changed and should repel each other if only electrostatic forces were involved. Preliminary experiments showed no binding of Factor IX to dextran sulphate Sepharose or SPSephadex at neutral pH further supporting the concept of specific binding of heparin to Factor IX. It is likely that both the charged sulphate groups and the carbohydrate structures of heparin are involved in the binding. The chemical characterization of pure Factor IX shows that it is a glycoprotein with a molecular weight of about 72 000 composed of a single peptide chain crosslinked by 13 disulphide bridges. The amino acid composition is that of normal globular protein but the fairly large proportion of hydrophilic amino acids should be noted. The amino acid composition is similar to that of human prothrombin (12) but some notable differences are found, especially the higher contents of cystine and lysine and the lower content of tyrosine in Factor IX as compared to prothrombin. The aminoterminal amino acids are also different being tyrosine for Factor IX and alanine for prothrombin. Comparison of bovine (11) and human Factor IX show that the amino acid compositions are very similar and there is also good agreement with respect to the carbohydrate content. The aminoterminal amino acid is also the same. There is however a considerable difference in molecular weight, 55 000 for the bovine factor and 72 000 for the human. Recently another method for purification of human Factor IX was published (13). The pure protein obtained showed about the same molecular weight as the material obtained in this study but there are important differences in other respects. The amino-terminal acids are different being tyrosine in this study and glycine in the other study. There are also significant differences in the amino and carbohydrate compositions. ACKNOWLEDGMENT Thanks are due to Dr Linda Fryklund for valuable discussions and for performing the aminoterminal analyses. Thanks are also due to Mrs Anita Linder for excellent technical assistance and to Mrs Karin Brundin for typing the manuscript.
vo1.7,40.3
HUMAN
FACTOR
Ix PURIFICATION
459
REFERENCES 1.
ANDERSSON,
L-O., BORG, H. and MILLER-ANDERSSON, M. Purification of Factor IX by Affinity Chromatography. Abstract from IVth International Congress on Thrombosis and HaemostasisT Wien, 1973, p 142.
2. VELTKAMP, J.J., DRION, E.F. and LOELIGER, E.A. Detection of the Carrier State in Hereditary Coagulation Disorders I. Thromb. Diath. Haemorrhag. 19-20, 279, 1968. 3. NOREN, I. Specific Assay of Prothrombin. Stand. J. Clin. Lab. Invest. 25, 47, 1970. 4. DENSON, K.W.E. The Specific Assay of Prower-Stuart Factor and Factor VII. Acta Haematologica 25, 105. 5. DAVIES, B.J. Disc. electrophoresis-II. Method and Application to Human Serum Proteins. Ann. N.Y. Acad. Sci.121, 404, 1964. 6. ANDERSSON, L-O., BORG, H. and MIKAELSSON, M. Molecular Weight Estimations of Protein by Electrophoresis in Polyacrylamide gels of Graded Porosity. FEBS Letters, 20, 199, 1972. 7. SCHEIDEGGER, J.J. Une Micromethode de L'immunoelectrophorese. Int Arch. Allergy. Appl. Irxaunol.7, 103, 1955. 8. FRYKLUND, L., EAKER, D. and KARLSSON, E. Amino Acid Sequences of the two Principal Neurotoxins of Enhydrina Scistosa Venom. Biochemistry, 11, 4633, 1972. 9. DUBOIS, M., GILLES, K.A., HAMILTON, J.K.. REBERS, P.A. and SMITH, F. Calorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 28, 350, 1956. 10. WARREN, L. The Thiobarbituric Acid Assay of Sialic Acids. J. Biol. Chem. 234, 1971, 1959. 11. FUJIKAWA, K., THOMPSON, A.R., LEGAZ, M.E., MEYER, R.G. and DAVIE, E.W. Isolation and Characterization of Bovine Factor IX. Biochemistry, 12, 4938, 1973. 12. KISIEL, W. and HANAHAN, D.J. Purification and Characterization of Human Factor II. Biochim, Biophys. Acta. 304, 103, 1973. 13. aSTERLiD,B. and FLENGSRUD, R. Purification and Some Characteristics of the Coagulation Factor IX from Human Plasma. Biochem. J. 145, 469, 1975.
