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O-phosphoserine as a constituent of polypeptide chains of bovine fibrinogen
174
BIOCHIMICA ET BIOPHYSICAACTA
BBA 36128 O - P H O S P H O S E R I N E AS A CONSTITUENT OF P O L Y P E P T I D E CHAINS Ol; BOVINE F I B R I N O G E N
T. K R A J E W S K I
AND CZ. C I E R N I E W S K [
Department of Biochemistry, Institute of Biochemistry and Physiology, Umvcrsi~y ,!f LSd.!, t.~;d~ (Poland) (Received J a n u a r y i o t h , 1972)
SUMMARY
The subunits chains obtained by sulphitolysis of bovine fibrinogen have been separated by column chromatography into four fractions; I, II, III and IV, corresponding to the y-I, y - 2 , fi(B) and a (A)* chains, respectively. From the last two fractions phosphoserine was isolated by thin-layer chromatography. In Fractions I and II only trace amounts of phosphorus were detected. The role of phosphorus in the conversion of fibrinogen to fibrin is discussed.
INTRODUCTION
The tertiary structure of the fibrinogen molecule is due to the disulphide bonds formed by 56 or 58 cysteins 1. After sulphitolysis of the disulphide bonds in fibrinogen, flee polypeptide chains appeared 2 7. This sulphitolysis product can be separated into a(A), fl(B) and ~ chains during column chromatography on CM- and DEAE-cellulosel,4,'~,7. It was thought previously that the fibrinogen molecule consisted of 3 pairs of identical polypeptide chains3, *,8. The investigations of the subunits obtained by sulphitolysis of fibrinogen in polyacrylamide gel electrophoresis have shown considerable differences between individual chains% This finding has been confirmed by amino acid and saccharide analyses of ~ chains. The investigations carried out by Claire et al. 7 and Brummel and Montgomery 9 indicated the appearance of heterogeneity within the remaining polypeptide chains. The phosphorus content of mammalian fibrinogen and fibrin was determined by Dmochowski and KrajewskP °, Fantl and Ward n, Blomb~tck et al. 1~ and Krajewski and Dmochowski 13. It was found that human and animal fibrinogen contained 1-2 phosphorus atoms/molecule. In the present paper the location of tlle phosphorus within the individual chains and possibly the character of the existing phosphorus compound have been elucidated. * T h e n o m e n c l a t u r e r e c o m m e n d e d b y t h e I n t e r n a t i o n a l C o m m i t t e e on H a e m o s t a s i s a n d T h r o n l b o s i s ( W a s h i n g t o n 1967) for t h e t h r e e p e p t i d e c h a i n s in fibrinogen is a (A), fl (B), a n d y.
Biochim. Biophys. Acta, 271 (1972) 174-181
O-PHOSPHOSERINE IN FIBRINOGEN
175
MATERIALS AND METHODS
Preparation of fibrinogen Fibrinogen was isolated from bovine citrated plasma by the ether precipitation technique of Kekwick et al. 1. and stored as an 0.5% solution in 0.3 M NaCI at --20 °C until required for use. The purified protein was 96% coagulable by thrombin. Only reagents of analytical grade were used. Preparation of S-sulphofibrinogen S-Sulphofibrinogen was prepared according to the sulphitolysis procedure of Henschen 3. Purified protein before using was separated from the reagents by dialysis at 4 °C for 24h against distilled water brought to pH 8. 5 with ammonia. I g of fibrinogen precipitated during this process was dissolved in 72 ml of 9 M urea solution and afterwards i4-ml portions of a freshly prepared saturated solution of sodium sulphite were gradually added with continuous stirring. The pH was maintained within the range of 8.0-9.0 by means of 2 M HC1 and a concentrated solution of NHiOH. Then 5 ml of i M CuS04 was introduced into the mixture. The whole procedure was performed at room temperature and pH was maintained as above. S-Sulphofibrinogen prepared in this way was purified by dialysis according to Pechere et al. 15 and then freeze-dried. The separation of the subunits of S-sulphofibrinogen Ion-exchange chromatography using a column of CM-cellulose (2.4 cm × I5 cm) was employed for this purpose according to the technique of McKee et al. 5. Activated resin prepared as described by Peterson and Sober TM was equilibrated with 0.005 M sodium acetate buffer, pH 5.3, containing 8 M urea. Then 40 mg of the S-sulphofibrinogen was disolved in 3.5 ml of the same buffer and dialysed for 12 h against IOO ml of 0.005 M acetate buffer; pH 5.3 at 4 °C. A purified solution of S-sulphofibrinogen was applied to a column of resin and separated. The column was developed by the linear gradient elution method using 400 ml of the buffers (0.005 M and o.I M sodimn acetate buffer), pH 5.3, containing 8 M urea. Fractions of 5 ml were collected by volume and a flow rate of 50-60 ml/h was employed. Protein content of the fractions was monitored by measuring the absorbance at 282 nm. In further investigations the separation of the subunit polypeptide chains was carried out using step-wise gradient method. For this purpose the column was filled up with the activated CM-cellulose (4 cm × 15 cm) and washed out with 0.03 M acetate buffer at pH 4.8. 800 mg of S-sulphofibrinogen was dissolved in IO ml of the same 0.03 M sodium acetate buffer, dialysed against ioo ml of the same buffer and then applied onto the previously prepared CM-cellulose column. The adsorbed compounds were successively eluted using sodium acetate buffers (pH 4.8, containing 8 M urea) in the following quantities: 200 ml of 0.03 M buffer, 500 ml of 0.05 M buffer, IOOOml of 0.075 M buffer and 500 ml of o.i M buffer. Fractions of io ml were collected by volume at room temperature. A flow rate of 30 ml/h was employed. The fractions giving individual column peaks were poured together and separately dialysed against distilled water brought to pH 8. 5 with ammonia.
Biochim. Biopkys. Hera, 27r (1972) I74-18I
176
T. KRAJEWSKI, CZ. CIERNIEWSKI
Determination of phosphorus The phosphorus content in dry substances of each fraction was deternfined according to a modifed Kutner-Lichtenstein method ~v. Combustion was made in concentrated H2SO 4 with addition of some drops of 60% HCIO a. The colour was developed strictly in medium 0. 5 M H2SO 4 (reduction of the phosphor m o l y b d a t e c(mlplex but not molybdate). Between I and IO #g P was determined by this method.
Acid hydrolysis Samples (2- 4 rag) of the same as the above material were partially hydrolysed in test tubes with 2 ml of 2 M HC1 at lO0 °C for IO tl. HC1 was removed in a vacuum desiccator over solid P20~ and K O H and the residues were investigated by thinlayer chromatography,
Two-dimensional thin-layer chromatography Silica gel (Silica gel DF 5, Comag Ag, Muttenz, Switzerland) was used. The thickness of the gel layer was o.25 ram. 15 #1 of I mM standard solution containing the following amino acids: serine, threonine, phosphoserine, phosphothreonine, aspartic acid, arginine and IO-/A samples of hydrolysate were applied to the glass platelets and separately subjected to chromatography. The following solvent systems were used : Solvent A : chloroformm e t h a n o l - I 7 % (v/v) NH4OH (40:4o:20, by vol.); Solvent B: phenol water (75 g phenol + 25 ml distilled water). Ascending chromatography was used. In the first dimension the solvent was run for 3 h and ill the second dimension fl)r IO h. After drying, the paper was developed with ninhydrin reagent.
Low-voltage electrophoresis-chromatography A conventional horizontal apparatus was used. 2o-/,1 samples of our hydrolysate and standard solutions of amino acids were applied to W h a t m a n No. I paper and subjected to electrophoresis. The runs were made in the following buffer systems: acetic acid-formic acid-water (2oo:60:740, by vol.), pH 1.5, at 500 V and 14 mA for 3 11. In the second dimension chromatography was employed using tile following solvent system: butanol-acetic acidwater (4:1:1.5, by vol.) for 12 h. In later investigations only electrophoresis as above was performed (width of Whatnlan No. I paper strips was 3-4 cm). RESULTS
Treatments of tile fibrinogen with sulfite in urea solution gave a complete conversion to the corresponding S-sulpho-derivatives. Sulphitolysis products were soluble in water at pH 8.5. Tile separation of this S-sulphofibrinogen into the polypeptide chains by means of column chromatography on CM-eellulose using a linear gradient according to McKee et al. 5 is presented on Fig. Ia. In order to obtain a more clear separation in the next experiments a step-wise gradient was employed according to Claire et al.L The example of S-sulphofibrinogen separation is shown in Fig. lb. The four fractions which were designated I, II, I I I and IV, respectively, were noticeable. In Table I results concerning the phosphorus content of fibrinogen, S-sulphofibrinogen and individual column peaks are assembled. I t was flmnd that S-sulphoBiochim. Biophys. Acta, 27I (1972) 174-181
17 7
0 - P H O S P H O S E R I N E IN FIBRINOGEN a
o.2
J O.!
) Errtuent ImO 2~-b
1
|
.i/ 2OO
Effluent (ml)
Fig. i. C h r o m a t o g r a m s o b t a i n e d b y t h e s e p a r a t i o n o f s u l p h i t o l y s e d fibrinogen in 8 M u r e a on CM-cellulose u s i n g linear g r a d i e n t (a) a n d step-wise g r a d i e n t (b). T h e detailed p r o c e d u r e is described in Material a n d Methods.
TABLE I THE PHOSPHORUS CONTENT OF BOVINE FIBRINOGEN, S-SULPHOFIBRINOGEN AND ISOLATED FRACTIONS I, II, I I I AND IV (see text)
Fraction
Phosphorus content (t*g/Ioo mg protein)
Mol. wt
mmoles P/mole protein
% Total amount of mmoles p
Fibrinogen S-sulphofibrinogen I (7-1 chain) II (7-2 chain) I I I (fl (B) chain) IV (a (A) chain)
17"9 17. 5 3.8 3.5 24. 5 29.8
334 332 47 47 56 63
1929 1874 58 53 443 61o
ioo 97.1 3.0 2.7 23.0 31.6
ooo ooo ooo ooo ooo 5o0
Biochim. Biophys. Acta, 271 (1972) 174_181
178
z. KRAJEWSK[, CZ. CIERNIEWSKI
Fig. 2. T w o - d i m c n t i o n a l t h i n - l a y e r c h r o i n a t o g r a p h y of residue from a partial h \ ' d r o l v s i s ~,f F r a c t i o n I i [ (a) a n d F r a c t i o n IV (b). Arrows indicate t h e directiou of miRration.
fibrinogen contained the same amount of phosphorus as native fibrinogen (17 I8 #gP/ IOO mg protein). Fractions I and I I showed only trace amounts of phosphorus. This means that nearly all the phosphorus is in Fractions I I I and IV. As shown in table I, Fraction I I I contained 24.5 #g P/IoO mg and Fraction IV 29.8 ~g P/IoO rag.
% .oK
O
0 ®
o
- -
SoLventB
Fig. 3- T w o - d i m e n t i o n a l t h i n - l a y e r c h r o m a t o g r a p h y of s t a n d a r d a m i n o acids : serine, O - p h o s p h o serine, t h r e o n i n e , O - p h o s p h o t h r e o n i n e , a s p a r t i c acid a n d arginine (I5-tA s a m p l e s of i - m m o l e s o l u t i o n s were applied).
13iochim. Biophys. dcta, 271 (1972) 174-181
0-PHOSPHOSERINE IN FIBRINOGEN
179
Samples of the phosphorus-rich fractions were hydrolysed with 2 M HC1 at ioo °C for I0 h and subjected to thin-layer chromatography and electrophoresis chromatography (see Materials and Methods). Fig. 2a presents the separation of hydrolysates of Fraction III by thin-layer chromatography after spraying with ninhydrin. The separation of Fraction IV hydrolysate in the same way gave an almost identical chromatography pattern (Fig. 2b). In parallel to this assay serine, O-phosphoserine, threonine and O-phosphothreonine were subjected to thin-layer chromatography in order to establish their characteristic positions on the platelets (Fig. 3). It is noticeable in Fig. 2 that as a result of the separation of our hydrolysate a spot in the position corresponding to
0
0
c) 0
o '
b
Qectrophorests
%
Fig. 4. Low-voltage paper electrophoresis-chromatography of residue from a partial hydrolysis of Fraction I I I (a) and standard amino acids (b).
Biochim. Biophys. Acta, 27i (1972) i74-z8z
I~0
T. KRAJEWSKI, CZ. CIERNIEWSKI
Fig. 5. L o w - v o l t a g e p a p e r elcctrophoresis p a t t e r n of residue f r o m a partial h y d r o l y s i s of Fraction I l l .
authentic phosphoserine appeared on the plate. A suggestion about the presence of the 0-phosphoserine in bovine fibrinogen was confirmed by the results obtained by electrophoresis-chromatography of hydrolysate of Fraction I I I (Fig. 4) and by electrophoresis (Fig. 5). The same results were obtained with Fraction IV. DISCUSSION
The location of the phosphorus in the fibrinogen molecule has not yet been established. It was found several years ago that a molecule of human or bovine fibrinogen contained I - 2 gatoms of organically bound phosphorus. In I962 Blomb~ick et al. 18,19 found that in respect to human fibrinogen approx. 3o-4o% of this phosphorus is being split off during the conversion of fibrinogen to fibrin by action of thrombin and can be found in fibrinopeptide A as a phosphoserine. On the contrary, when bovine fibrinogen is subjected to the action of thrombin the phosphorus content in f b r i n monomers does not change but rather the ester sulphate group is split off together with fibrinopeptide B 2°,2x. Biochim. Biophys. Acla, 27~ (i972) i74 ISI
0-PHOSPHOSEI~INE IN FIBRINOGEN
181
The subject of the present paper was to elucidate whether the phosphorus contained in the fibrinogen molecule is equally divided among three chains a(A),/~(B), y, or is limited only to some chains. We found that bovine fibrinogen subjected to sulphitolysis according to Henschen a contained the same amount of phosphorus as native fibrinogen; it means I7-I8 #g P/Ioo mg protein. After the separation of Ssulphofibrinogen into its individuai Fractions I, II, III and IV by column chromatography it was shown that almost the whole phosphorus is equally limited to Fractions III and IV corresponding to the ~(B) and a(A) chains, respectively. Fractions I and II (y-I, y-2, respectively) contained only trace amounts of phosphorus (Table I). The influence of phosphorus found in fibrinogen on its clottability is not yet clear. Blomb~ick et al. 1~ indicated that phosphorus-free fibrinogen reacts with thrombin in a similar way to native fibrinogen and can be quantitatively transformed to fibrin. However, a significant increase in clotting time was observed. According to the hypothesis of Witt and Mfiller 22 given in their recent communication the clottingtime depends on the phosphorus content of fibrinogen. The authors have also observed a significant prolongation of the clotting-time for dephosphorized human fibrinogen. It is quite possible that this is connected with the presence of the phosphate group which was found in the/~(B) and a(A) chains. During our previous preliminary investigations which are continued here, the presence of phosphoserine in tryptic digest fibrinogen was found (T. Krajewski and Cz. Cierniewski, unpublished results). After hydrolysis of Fractions III and IV and their separation by thin-layer chromatography it was established that both fl(B) and a(A) chains contained whole phosphoserine in almost the same amounts. On the basis of the observations of Witt and Miiller ~2 and our own results we came out with a suggestion that phosphorylation of serine in the a(A) and/~(B) chains has some influence on the conversion of fibrinogen into fibrin by the action of thrombin. REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22
A. H e n s c h e n , Ark. Kern., 22 (1964) 355. A. Henschen, Acta Chem, Scand., 16 (1962) 93A. H e n s c h e n , Ark. Kem., 22 (1963) I. J. Clegg and I. Bailey, Biochim. Biophys. Acta, 63 (1962) 525 . P. A. McKee, L. A. Rogers, E. Marler a n d R. L. Hill, Arch. Biochem. Biophys., 116 (1966) 271. S. I w a n a g a , A. Henschen and B. BlombAck, Acta Chem. Scand., 2o (1966) 1183. H. Claire, T. Gerberck, T. Yoshikawa and R. Montgomery, Biockim. Biophys. Acta, 134 (1969) 67 . B. Blomb/ick a n d I. Y a m a s h i n a , Ark. I4em., 12 (1958) 299. M. C. B r u m m e l a n d R. M o n t g o m e r y , Anal. Biochem., 33 (197 o) 28. A. Dmochowski and T. Krajewski, Proc. 5th Int. Congr. Biochem., Moscow, I96I, Section 16 p. 348. P, Fantl and H. A. W a r d , Biochim. Biophys. Acta, 64 (1962) 568. B. BlombAck, M. Blomb/ick a n d J. Searle, Biochim. Biophys. dcta, 74 (1963) 148. T. Krajewski and A. Dmochowski, Lddz. Towarz. Nauk., Wydziat III, No. 95, 1963. R. A. Kekwick, M. E. Mc K a y , M. H. Nance and B. R. Record, Biochem. J., 6o (1955) 671. J. F. Pechere, G. H. Dixon, R. Maybury and H. Neurath, J. Biol. Chem., 233 (1958) 1394. E. A. Peterson and H. Sober, J. Am. Chem. Soc., 78 (1956) 751. A. D m o c h o w s k i , T. Krajewski and H. Urbanek, Chem. Anal., 5 (196o) 683. B. Blomb~ick, M. B l o m b ~ c k , P. Edman and B. Hessel, Nature, 193 (1962) 883. B. Blomb/ick, P. Edman and B. Hessel, Biochim. Biophys. Acta, 115 (1966) 371. F. R. B e t t e l h e i m , Biochim. Biophys. Acta, 19 (1956) 121. T. Krajewski and B. Blombiick, Acta Chem. Scand., 22 (1968) 1339. I. Witt and H. Mfiller, Biochim. Biophys. Acta, 221 (197 o) 402.
Biochim. Biophys. Acta, 271 (1972) I74-181
BIOCHIMICA ET BIOPHYSICAACTA
BBA 36128 O - P H O S P H O S E R I N E AS A CONSTITUENT OF P O L Y P E P T I D E CHAINS Ol; BOVINE F I B R I N O G E N
T. K R A J E W S K I
AND CZ. C I E R N I E W S K [
Department of Biochemistry, Institute of Biochemistry and Physiology, Umvcrsi~y ,!f LSd.!, t.~;d~ (Poland) (Received J a n u a r y i o t h , 1972)
SUMMARY
The subunits chains obtained by sulphitolysis of bovine fibrinogen have been separated by column chromatography into four fractions; I, II, III and IV, corresponding to the y-I, y - 2 , fi(B) and a (A)* chains, respectively. From the last two fractions phosphoserine was isolated by thin-layer chromatography. In Fractions I and II only trace amounts of phosphorus were detected. The role of phosphorus in the conversion of fibrinogen to fibrin is discussed.
INTRODUCTION
The tertiary structure of the fibrinogen molecule is due to the disulphide bonds formed by 56 or 58 cysteins 1. After sulphitolysis of the disulphide bonds in fibrinogen, flee polypeptide chains appeared 2 7. This sulphitolysis product can be separated into a(A), fl(B) and ~ chains during column chromatography on CM- and DEAE-cellulosel,4,'~,7. It was thought previously that the fibrinogen molecule consisted of 3 pairs of identical polypeptide chains3, *,8. The investigations of the subunits obtained by sulphitolysis of fibrinogen in polyacrylamide gel electrophoresis have shown considerable differences between individual chains% This finding has been confirmed by amino acid and saccharide analyses of ~ chains. The investigations carried out by Claire et al. 7 and Brummel and Montgomery 9 indicated the appearance of heterogeneity within the remaining polypeptide chains. The phosphorus content of mammalian fibrinogen and fibrin was determined by Dmochowski and KrajewskP °, Fantl and Ward n, Blomb~tck et al. 1~ and Krajewski and Dmochowski 13. It was found that human and animal fibrinogen contained 1-2 phosphorus atoms/molecule. In the present paper the location of tlle phosphorus within the individual chains and possibly the character of the existing phosphorus compound have been elucidated. * T h e n o m e n c l a t u r e r e c o m m e n d e d b y t h e I n t e r n a t i o n a l C o m m i t t e e on H a e m o s t a s i s a n d T h r o n l b o s i s ( W a s h i n g t o n 1967) for t h e t h r e e p e p t i d e c h a i n s in fibrinogen is a (A), fl (B), a n d y.
Biochim. Biophys. Acta, 271 (1972) 174-181
O-PHOSPHOSERINE IN FIBRINOGEN
175
MATERIALS AND METHODS
Preparation of fibrinogen Fibrinogen was isolated from bovine citrated plasma by the ether precipitation technique of Kekwick et al. 1. and stored as an 0.5% solution in 0.3 M NaCI at --20 °C until required for use. The purified protein was 96% coagulable by thrombin. Only reagents of analytical grade were used. Preparation of S-sulphofibrinogen S-Sulphofibrinogen was prepared according to the sulphitolysis procedure of Henschen 3. Purified protein before using was separated from the reagents by dialysis at 4 °C for 24h against distilled water brought to pH 8. 5 with ammonia. I g of fibrinogen precipitated during this process was dissolved in 72 ml of 9 M urea solution and afterwards i4-ml portions of a freshly prepared saturated solution of sodium sulphite were gradually added with continuous stirring. The pH was maintained within the range of 8.0-9.0 by means of 2 M HC1 and a concentrated solution of NHiOH. Then 5 ml of i M CuS04 was introduced into the mixture. The whole procedure was performed at room temperature and pH was maintained as above. S-Sulphofibrinogen prepared in this way was purified by dialysis according to Pechere et al. 15 and then freeze-dried. The separation of the subunits of S-sulphofibrinogen Ion-exchange chromatography using a column of CM-cellulose (2.4 cm × I5 cm) was employed for this purpose according to the technique of McKee et al. 5. Activated resin prepared as described by Peterson and Sober TM was equilibrated with 0.005 M sodium acetate buffer, pH 5.3, containing 8 M urea. Then 40 mg of the S-sulphofibrinogen was disolved in 3.5 ml of the same buffer and dialysed for 12 h against IOO ml of 0.005 M acetate buffer; pH 5.3 at 4 °C. A purified solution of S-sulphofibrinogen was applied to a column of resin and separated. The column was developed by the linear gradient elution method using 400 ml of the buffers (0.005 M and o.I M sodimn acetate buffer), pH 5.3, containing 8 M urea. Fractions of 5 ml were collected by volume and a flow rate of 50-60 ml/h was employed. Protein content of the fractions was monitored by measuring the absorbance at 282 nm. In further investigations the separation of the subunit polypeptide chains was carried out using step-wise gradient method. For this purpose the column was filled up with the activated CM-cellulose (4 cm × 15 cm) and washed out with 0.03 M acetate buffer at pH 4.8. 800 mg of S-sulphofibrinogen was dissolved in IO ml of the same 0.03 M sodium acetate buffer, dialysed against ioo ml of the same buffer and then applied onto the previously prepared CM-cellulose column. The adsorbed compounds were successively eluted using sodium acetate buffers (pH 4.8, containing 8 M urea) in the following quantities: 200 ml of 0.03 M buffer, 500 ml of 0.05 M buffer, IOOOml of 0.075 M buffer and 500 ml of o.i M buffer. Fractions of io ml were collected by volume at room temperature. A flow rate of 30 ml/h was employed. The fractions giving individual column peaks were poured together and separately dialysed against distilled water brought to pH 8. 5 with ammonia.
Biochim. Biopkys. Hera, 27r (1972) I74-18I
176
T. KRAJEWSKI, CZ. CIERNIEWSKI
Determination of phosphorus The phosphorus content in dry substances of each fraction was deternfined according to a modifed Kutner-Lichtenstein method ~v. Combustion was made in concentrated H2SO 4 with addition of some drops of 60% HCIO a. The colour was developed strictly in medium 0. 5 M H2SO 4 (reduction of the phosphor m o l y b d a t e c(mlplex but not molybdate). Between I and IO #g P was determined by this method.
Acid hydrolysis Samples (2- 4 rag) of the same as the above material were partially hydrolysed in test tubes with 2 ml of 2 M HC1 at lO0 °C for IO tl. HC1 was removed in a vacuum desiccator over solid P20~ and K O H and the residues were investigated by thinlayer chromatography,
Two-dimensional thin-layer chromatography Silica gel (Silica gel DF 5, Comag Ag, Muttenz, Switzerland) was used. The thickness of the gel layer was o.25 ram. 15 #1 of I mM standard solution containing the following amino acids: serine, threonine, phosphoserine, phosphothreonine, aspartic acid, arginine and IO-/A samples of hydrolysate were applied to the glass platelets and separately subjected to chromatography. The following solvent systems were used : Solvent A : chloroformm e t h a n o l - I 7 % (v/v) NH4OH (40:4o:20, by vol.); Solvent B: phenol water (75 g phenol + 25 ml distilled water). Ascending chromatography was used. In the first dimension the solvent was run for 3 h and ill the second dimension fl)r IO h. After drying, the paper was developed with ninhydrin reagent.
Low-voltage electrophoresis-chromatography A conventional horizontal apparatus was used. 2o-/,1 samples of our hydrolysate and standard solutions of amino acids were applied to W h a t m a n No. I paper and subjected to electrophoresis. The runs were made in the following buffer systems: acetic acid-formic acid-water (2oo:60:740, by vol.), pH 1.5, at 500 V and 14 mA for 3 11. In the second dimension chromatography was employed using tile following solvent system: butanol-acetic acidwater (4:1:1.5, by vol.) for 12 h. In later investigations only electrophoresis as above was performed (width of Whatnlan No. I paper strips was 3-4 cm). RESULTS
Treatments of tile fibrinogen with sulfite in urea solution gave a complete conversion to the corresponding S-sulpho-derivatives. Sulphitolysis products were soluble in water at pH 8.5. Tile separation of this S-sulphofibrinogen into the polypeptide chains by means of column chromatography on CM-eellulose using a linear gradient according to McKee et al. 5 is presented on Fig. Ia. In order to obtain a more clear separation in the next experiments a step-wise gradient was employed according to Claire et al.L The example of S-sulphofibrinogen separation is shown in Fig. lb. The four fractions which were designated I, II, I I I and IV, respectively, were noticeable. In Table I results concerning the phosphorus content of fibrinogen, S-sulphofibrinogen and individual column peaks are assembled. I t was flmnd that S-sulphoBiochim. Biophys. Acta, 27I (1972) 174-181
17 7
0 - P H O S P H O S E R I N E IN FIBRINOGEN a
o.2
J O.!
) Errtuent ImO 2~-b
1
|
.i/ 2OO
Effluent (ml)
Fig. i. C h r o m a t o g r a m s o b t a i n e d b y t h e s e p a r a t i o n o f s u l p h i t o l y s e d fibrinogen in 8 M u r e a on CM-cellulose u s i n g linear g r a d i e n t (a) a n d step-wise g r a d i e n t (b). T h e detailed p r o c e d u r e is described in Material a n d Methods.
TABLE I THE PHOSPHORUS CONTENT OF BOVINE FIBRINOGEN, S-SULPHOFIBRINOGEN AND ISOLATED FRACTIONS I, II, I I I AND IV (see text)
Fraction
Phosphorus content (t*g/Ioo mg protein)
Mol. wt
mmoles P/mole protein
% Total amount of mmoles p
Fibrinogen S-sulphofibrinogen I (7-1 chain) II (7-2 chain) I I I (fl (B) chain) IV (a (A) chain)
17"9 17. 5 3.8 3.5 24. 5 29.8
334 332 47 47 56 63
1929 1874 58 53 443 61o
ioo 97.1 3.0 2.7 23.0 31.6
ooo ooo ooo ooo ooo 5o0
Biochim. Biophys. Acta, 271 (1972) 174_181
178
z. KRAJEWSK[, CZ. CIERNIEWSKI
Fig. 2. T w o - d i m c n t i o n a l t h i n - l a y e r c h r o i n a t o g r a p h y of residue from a partial h \ ' d r o l v s i s ~,f F r a c t i o n I i [ (a) a n d F r a c t i o n IV (b). Arrows indicate t h e directiou of miRration.
fibrinogen contained the same amount of phosphorus as native fibrinogen (17 I8 #gP/ IOO mg protein). Fractions I and I I showed only trace amounts of phosphorus. This means that nearly all the phosphorus is in Fractions I I I and IV. As shown in table I, Fraction I I I contained 24.5 #g P/IoO mg and Fraction IV 29.8 ~g P/IoO rag.
% .oK
O
0 ®
o
- -
SoLventB
Fig. 3- T w o - d i m e n t i o n a l t h i n - l a y e r c h r o m a t o g r a p h y of s t a n d a r d a m i n o acids : serine, O - p h o s p h o serine, t h r e o n i n e , O - p h o s p h o t h r e o n i n e , a s p a r t i c acid a n d arginine (I5-tA s a m p l e s of i - m m o l e s o l u t i o n s were applied).
13iochim. Biophys. dcta, 271 (1972) 174-181
0-PHOSPHOSERINE IN FIBRINOGEN
179
Samples of the phosphorus-rich fractions were hydrolysed with 2 M HC1 at ioo °C for I0 h and subjected to thin-layer chromatography and electrophoresis chromatography (see Materials and Methods). Fig. 2a presents the separation of hydrolysates of Fraction III by thin-layer chromatography after spraying with ninhydrin. The separation of Fraction IV hydrolysate in the same way gave an almost identical chromatography pattern (Fig. 2b). In parallel to this assay serine, O-phosphoserine, threonine and O-phosphothreonine were subjected to thin-layer chromatography in order to establish their characteristic positions on the platelets (Fig. 3). It is noticeable in Fig. 2 that as a result of the separation of our hydrolysate a spot in the position corresponding to
0
0
c) 0
o '
b
Qectrophorests
%
Fig. 4. Low-voltage paper electrophoresis-chromatography of residue from a partial hydrolysis of Fraction I I I (a) and standard amino acids (b).
Biochim. Biophys. Acta, 27i (1972) i74-z8z
I~0
T. KRAJEWSKI, CZ. CIERNIEWSKI
Fig. 5. L o w - v o l t a g e p a p e r elcctrophoresis p a t t e r n of residue f r o m a partial h y d r o l y s i s of Fraction I l l .
authentic phosphoserine appeared on the plate. A suggestion about the presence of the 0-phosphoserine in bovine fibrinogen was confirmed by the results obtained by electrophoresis-chromatography of hydrolysate of Fraction I I I (Fig. 4) and by electrophoresis (Fig. 5). The same results were obtained with Fraction IV. DISCUSSION
The location of the phosphorus in the fibrinogen molecule has not yet been established. It was found several years ago that a molecule of human or bovine fibrinogen contained I - 2 gatoms of organically bound phosphorus. In I962 Blomb~ick et al. 18,19 found that in respect to human fibrinogen approx. 3o-4o% of this phosphorus is being split off during the conversion of fibrinogen to fibrin by action of thrombin and can be found in fibrinopeptide A as a phosphoserine. On the contrary, when bovine fibrinogen is subjected to the action of thrombin the phosphorus content in f b r i n monomers does not change but rather the ester sulphate group is split off together with fibrinopeptide B 2°,2x. Biochim. Biophys. Acla, 27~ (i972) i74 ISI
0-PHOSPHOSEI~INE IN FIBRINOGEN
181
The subject of the present paper was to elucidate whether the phosphorus contained in the fibrinogen molecule is equally divided among three chains a(A),/~(B), y, or is limited only to some chains. We found that bovine fibrinogen subjected to sulphitolysis according to Henschen a contained the same amount of phosphorus as native fibrinogen; it means I7-I8 #g P/Ioo mg protein. After the separation of Ssulphofibrinogen into its individuai Fractions I, II, III and IV by column chromatography it was shown that almost the whole phosphorus is equally limited to Fractions III and IV corresponding to the ~(B) and a(A) chains, respectively. Fractions I and II (y-I, y-2, respectively) contained only trace amounts of phosphorus (Table I). The influence of phosphorus found in fibrinogen on its clottability is not yet clear. Blomb~ick et al. 1~ indicated that phosphorus-free fibrinogen reacts with thrombin in a similar way to native fibrinogen and can be quantitatively transformed to fibrin. However, a significant increase in clotting time was observed. According to the hypothesis of Witt and Mfiller 22 given in their recent communication the clottingtime depends on the phosphorus content of fibrinogen. The authors have also observed a significant prolongation of the clotting-time for dephosphorized human fibrinogen. It is quite possible that this is connected with the presence of the phosphate group which was found in the/~(B) and a(A) chains. During our previous preliminary investigations which are continued here, the presence of phosphoserine in tryptic digest fibrinogen was found (T. Krajewski and Cz. Cierniewski, unpublished results). After hydrolysis of Fractions III and IV and their separation by thin-layer chromatography it was established that both fl(B) and a(A) chains contained whole phosphoserine in almost the same amounts. On the basis of the observations of Witt and Miiller ~2 and our own results we came out with a suggestion that phosphorylation of serine in the a(A) and/~(B) chains has some influence on the conversion of fibrinogen into fibrin by the action of thrombin. REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22
A. H e n s c h e n , Ark. Kern., 22 (1964) 355. A. Henschen, Acta Chem, Scand., 16 (1962) 93A. H e n s c h e n , Ark. Kem., 22 (1963) I. J. Clegg and I. Bailey, Biochim. Biophys. Acta, 63 (1962) 525 . P. A. McKee, L. A. Rogers, E. Marler a n d R. L. Hill, Arch. Biochem. Biophys., 116 (1966) 271. S. I w a n a g a , A. Henschen and B. BlombAck, Acta Chem. Scand., 2o (1966) 1183. H. Claire, T. Gerberck, T. Yoshikawa and R. Montgomery, Biockim. Biophys. Acta, 134 (1969) 67 . B. Blomb/ick a n d I. Y a m a s h i n a , Ark. I4em., 12 (1958) 299. M. C. B r u m m e l a n d R. M o n t g o m e r y , Anal. Biochem., 33 (197 o) 28. A. Dmochowski and T. Krajewski, Proc. 5th Int. Congr. Biochem., Moscow, I96I, Section 16 p. 348. P, Fantl and H. A. W a r d , Biochim. Biophys. Acta, 64 (1962) 568. B. BlombAck, M. Blomb/ick a n d J. Searle, Biochim. Biophys. dcta, 74 (1963) 148. T. Krajewski and A. Dmochowski, Lddz. Towarz. Nauk., Wydziat III, No. 95, 1963. R. A. Kekwick, M. E. Mc K a y , M. H. Nance and B. R. Record, Biochem. J., 6o (1955) 671. J. F. Pechere, G. H. Dixon, R. Maybury and H. Neurath, J. Biol. Chem., 233 (1958) 1394. E. A. Peterson and H. Sober, J. Am. Chem. Soc., 78 (1956) 751. A. D m o c h o w s k i , T. Krajewski and H. Urbanek, Chem. Anal., 5 (196o) 683. B. Blomb~ick, M. B l o m b ~ c k , P. Edman and B. Hessel, Nature, 193 (1962) 883. B. Blomb/ick, P. Edman and B. Hessel, Biochim. Biophys. Acta, 115 (1966) 371. F. R. B e t t e l h e i m , Biochim. Biophys. Acta, 19 (1956) 121. T. Krajewski and B. Blombiick, Acta Chem. Scand., 22 (1968) 1339. I. Witt and H. Mfiller, Biochim. Biophys. Acta, 221 (197 o) 402.
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