718
BIOCHEMICAL SOCIETY TRANSACTIONS
The Interaction of Clotting Factor X with Adsorbents Containing Bivalent Cations ROY M. HOWELL and SUSAN L. M. DEACON Department of Biochemistry, Queen Elizabeth College, London W8 7AH, U.K.
The prothrombin group of coagulant proteins contains adjacent pairs of y-carboxylated glutamic acid residues (Stenflo et al., 1974), which gives them the ability to chelate bivalent cations. This accounts for the specific adsorption of factors 11, VII, IX and X to BaSO, and bariumcitrate. Affinity for Ca2+ions also plays a role in adsorption to calcium phosphate, but, according to Tiselius et al. (1956), other factors, such as a variable chemical composition, contribute and make this adsorbent less specific than BaS0, for clotting-factor protein. In previous studies, Howell & Deacon (1975) established that the porous nature of particles of low-density BaSO,, compared with the solid crystalline appearance of other grades, accounted not only for its lower bulk density but also for its greater surface/ weight ratio and an ability to adsorb more protein from serum. Our interest in defining the physical and chemical properties of adsorbent particles is also concerned with the ability of some surfaces, such as those provided by high-density crystalline BaSO,, to catalyse the activation of the adsorbed clotting enzyme, factor X (Howell & Dupe, 1972). As a first step, more information was required on the amount of serum protein adsorbed to the complete range of adsorbents and the preparation of adsorbable protein from a sample ofeach material was carried out by the procedure of Howell &Scott (1964) and determined by the method of Lowry et a/. (1951). Since the four members of the prothrombin group are closely related and adsorbed together, we monitored their uptake and any activating ability of a surface by measuring the total amount of factor X, as well as the proportion of pre-formed factor Xa activity by the differential assay described previously (Howell & Dupe, 1972). This factor was also chosen as an example of the group because it occupies a central place in the pathways leading to prothrombin activation, and owing to this position, it may be involved in the generation of a thrombotic state. The wide divergence in the specific amount of protein and factor X adsorbed by the insoluble precipitates based on barium and calcium is shown in Tables 1 and 2 respectively. InTable 1, theability ofhigh-density grades of BaSO, to activate factor Xconfirms earlier findings (Howell &Dupe, 1972). The greater efficiency of BaS0, as an adsorbent over barium citrate was unexpected and could lead to a reappraisal of this more recent adsorbent for factor X. Barium citrate is normally prepared in situ and the yield/g was 5-10 times less than for BaS0,. The lower specificity of all forms of calcium phosphate for factor X, compared with BaSO,, is shown in Table 2. Since a much higher proportion of the protein adsorbed on the BaS0, samples represented clotting-factor protein, calcium phosphate was excluded from the next stage of the investigation. This involved an examination of the electrophoretic mobilities of serum protein adsorbed on particles of high- and low-density grades of BaSO,, as well as barium citrate, over the pH range 2-1 1. Measurements were made at 25°C in 0.02Mbarbitone buffer as described by Howell &Deacon (1975). This method has been used by Chattoraj & Bull (1959) to detect changes in adsorbed molecules which are not easily revealed by other means, and if the underlying surface were to influence the electrophoretic mobility of adsorbed clotting-factor protein, it could help to explain factor X activation. The pH-mobility curve of protein adsorbed on an inert particle should not differ greatly from that of dissolved protein, and a typical ‘titration curve’ was obtained with particles of low-density forms of BaSO, coated with serum protein. However, the pHmobility curve for the high-density grade was only superimposable on the former over 1976
P
Table 1. Uptake of protein and factor X from pig serum by barium citrate and various grades of BaSO, at p Each adsorbent (500mg) was added in turn to samples of serum (6ml). Protein was eluted from BaS04 sam (2.5 ml) and from barium citrate with 35 % (w/v) (NH,),SO,, pH7.2 (2.5 ml). Other conditions, as well as the previously (Howell &Dupe, 1972). One unit of factor X-clotting activity is taken as the amount present in each adsorbent is given in parentheses. -, Not determined. Commercial barium citrate (I.C.N. ;K and K Kodak, Kirkby, Liverpool, U.K., and the precipitated form was prepared in situ as described by Tishkoff Howell & Dupe (1972). Adsorbent Low-density BaSO, (0.8 g/ml) High-density BaS04 (1.7g/ml) Natural barytes (2.8 g/ml) Commercial barium citrate (0.9g/ml) Precipitated barium citrate (-)
Protein eluted Olg/mg of adsorbent) 7.8 2.4 2.0 3.0 6.1
Total factor X activity (units/g of adsorbent)
10' x Sp (units/m
3.3 1.2 1.5 0.003 0.4
Table 2. Uptake of protein and factor X from pig serum by various forms of calcium phosphate Details as in Table 1 , except that the protein was eluted with 0.25~-potassiumphosphate buffer, pH7.0, a adsorbents. Hydroxyapatite was prepared by the method of Tiselius et al. (1956). Calcium phosphate gel Poole, Dorset, U.K. Adsorbent Hydroxyapatite Calcium phosphate gel Calcium orthophosphate
Protein eluted (pglrng of adsorbent) 99 298 37
Total factor X activity (units/g of adsorbent) 4.7 8.5 0.8
lo2 x Sp (units/m
720
BIOCHEMICAL SOCIETY TRANSACTIONS
the pH range 2-6. From pH6 to 10.2 the mobilities of the protein-coated high-density particles accelerated to a level 35 % greater, at pH8.2, than the low-density particles. It is difficult at this stage to provide a complete explanation of this electrophoretic behaviour, but we consider it likely that on the smooth flat faces of the high-density particles, the protein is adsorbed as an expanded monolayer and allows a higher particle mobility from pH6 to 10.2, owing to the exposure of charged side groups (Chattoraj & Bull, 1959). In contrast, the uptake of adsorbed protein within the porous aggregates of low-density BaSOI (Howell & Deacon, 1975) prevents the increased mobility owing to unfolding of the protein. If this interpretation is valid, it would support the proposal of Howell & Dupe (1972) that partial unfolding on this adsorbent encourages autoproteolysis of factor X leading to its activation. For protein adsorbed to particles of barium citrate the pH-mobility curve showed a slight displacement in the acid region compared with the bare particles and reflects the small amount of protein adsorbed. The pH-mobility curve obtained for barium citrate precipitated in situ was interesting in that the linear variation of mobility with pH is typical of non-ionogenic particles (Goddard, 1970). This supports the proposal of Tishkoff et al. (1968) that the protein is sequestered within a crystal lattice, and in this state, it is unlikely that any unfolding and activation can occur. S. L. M. D. acknowledges receipt of a Science Research Council studentship.
Chattoraj, D. K. & Bull, H. B. (1959) J. Am. Chem. SOC.81, 5128-5136 Goddard, G. H. (1970) Ph.D. Thesis, University of London Howell, R. M. & Deacon, S. L. M. (1975) Thromb. Diarh. Haemorrh. 33, 256-270 Howell, R. M. & Dupe, R. J. (1972) Thromb. Diath.Haemorrh. 28, 306-316 Howell, R. M. & Scott, G . B. D. (1964) Br. J. Exp. Pathol. 45, 618-626 Lowry,O. H., Rosebrough,N.J., Farr, A. L. &Randall, R. J. (1951)J.Biol.Chem. 193,265-275 Stenflo, J., Fernlund, P., Egan, W. & Roepstorff, P. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 273C-2733 Tiselius, A., HjCrten, S. 0. & Levin, 6.(1956) Arch. Biochem. Biophys. 65, 132-155 Tishkoff,G . H., Williams, L. C. & Brown, D. M. (1968) J. Biol. Chem. 243,4151-4167
Physical Studies on Calcium Phosphate and Barium Citrate Used as Clotting-Factor Adsorbents SUSAN L. M. DEACON and ROY M. HOWELL Department of Biochemistry, Queen Elizabeth College, London W8 7AH, U.K.
Inorganic precipitates, containing Ca2+or BaZ+cations, have been used extensively in purification procedures for removing the vitamin K-dependent clotting factors from serum or plasma. The prothrombin group, which consists of factors 11, VII, IX and X, contains the modified amino acid y-carboxyglutamic acid which is responsible for the high cation-binding capacity of these proteins (Stenflo et al., 1974). We have examined the crystalline structure and charge of these inorganic compounds more closely, by scanning electron microscopy and particle electrophoresis, in a n attempt to relate physical properties and chemical composition to their properties as clotting-factor adsorbents. The present investigation on the physical properties of calciumphosphate and bariumcitrate is similar to our previous study of BaS04asaclottingfactor adsorbent (Howell & Deacon, 1975). The electrophoretic mobility of particles of various adsorbents was measured at 25°C in 0.02~-barbitonebuffer over the pH range 2-1 1 by the procedure of Bangham et al. (1958). Despite differences in morphology, various grades of BaSO,, in the former study, gave similar pH-mobility curves, since the internal composition of the crystal remains relatively constant. Incontrast, the present study showed a considerable shift in theshape 1976
BIOCHEMICAL SOCIETY TRANSACTIONS
The Interaction of Clotting Factor X with Adsorbents Containing Bivalent Cations ROY M. HOWELL and SUSAN L. M. DEACON Department of Biochemistry, Queen Elizabeth College, London W8 7AH, U.K.
The prothrombin group of coagulant proteins contains adjacent pairs of y-carboxylated glutamic acid residues (Stenflo et al., 1974), which gives them the ability to chelate bivalent cations. This accounts for the specific adsorption of factors 11, VII, IX and X to BaSO, and bariumcitrate. Affinity for Ca2+ions also plays a role in adsorption to calcium phosphate, but, according to Tiselius et al. (1956), other factors, such as a variable chemical composition, contribute and make this adsorbent less specific than BaS0, for clotting-factor protein. In previous studies, Howell & Deacon (1975) established that the porous nature of particles of low-density BaSO,, compared with the solid crystalline appearance of other grades, accounted not only for its lower bulk density but also for its greater surface/ weight ratio and an ability to adsorb more protein from serum. Our interest in defining the physical and chemical properties of adsorbent particles is also concerned with the ability of some surfaces, such as those provided by high-density crystalline BaSO,, to catalyse the activation of the adsorbed clotting enzyme, factor X (Howell & Dupe, 1972). As a first step, more information was required on the amount of serum protein adsorbed to the complete range of adsorbents and the preparation of adsorbable protein from a sample ofeach material was carried out by the procedure of Howell &Scott (1964) and determined by the method of Lowry et a/. (1951). Since the four members of the prothrombin group are closely related and adsorbed together, we monitored their uptake and any activating ability of a surface by measuring the total amount of factor X, as well as the proportion of pre-formed factor Xa activity by the differential assay described previously (Howell & Dupe, 1972). This factor was also chosen as an example of the group because it occupies a central place in the pathways leading to prothrombin activation, and owing to this position, it may be involved in the generation of a thrombotic state. The wide divergence in the specific amount of protein and factor X adsorbed by the insoluble precipitates based on barium and calcium is shown in Tables 1 and 2 respectively. InTable 1, theability ofhigh-density grades of BaSO, to activate factor Xconfirms earlier findings (Howell &Dupe, 1972). The greater efficiency of BaS0, as an adsorbent over barium citrate was unexpected and could lead to a reappraisal of this more recent adsorbent for factor X. Barium citrate is normally prepared in situ and the yield/g was 5-10 times less than for BaS0,. The lower specificity of all forms of calcium phosphate for factor X, compared with BaSO,, is shown in Table 2. Since a much higher proportion of the protein adsorbed on the BaS0, samples represented clotting-factor protein, calcium phosphate was excluded from the next stage of the investigation. This involved an examination of the electrophoretic mobilities of serum protein adsorbed on particles of high- and low-density grades of BaSO,, as well as barium citrate, over the pH range 2-1 1. Measurements were made at 25°C in 0.02Mbarbitone buffer as described by Howell &Deacon (1975). This method has been used by Chattoraj & Bull (1959) to detect changes in adsorbed molecules which are not easily revealed by other means, and if the underlying surface were to influence the electrophoretic mobility of adsorbed clotting-factor protein, it could help to explain factor X activation. The pH-mobility curve of protein adsorbed on an inert particle should not differ greatly from that of dissolved protein, and a typical ‘titration curve’ was obtained with particles of low-density forms of BaSO, coated with serum protein. However, the pHmobility curve for the high-density grade was only superimposable on the former over 1976
P
Table 1. Uptake of protein and factor X from pig serum by barium citrate and various grades of BaSO, at p Each adsorbent (500mg) was added in turn to samples of serum (6ml). Protein was eluted from BaS04 sam (2.5 ml) and from barium citrate with 35 % (w/v) (NH,),SO,, pH7.2 (2.5 ml). Other conditions, as well as the previously (Howell &Dupe, 1972). One unit of factor X-clotting activity is taken as the amount present in each adsorbent is given in parentheses. -, Not determined. Commercial barium citrate (I.C.N. ;K and K Kodak, Kirkby, Liverpool, U.K., and the precipitated form was prepared in situ as described by Tishkoff Howell & Dupe (1972). Adsorbent Low-density BaSO, (0.8 g/ml) High-density BaS04 (1.7g/ml) Natural barytes (2.8 g/ml) Commercial barium citrate (0.9g/ml) Precipitated barium citrate (-)
Protein eluted Olg/mg of adsorbent) 7.8 2.4 2.0 3.0 6.1
Total factor X activity (units/g of adsorbent)
10' x Sp (units/m
3.3 1.2 1.5 0.003 0.4
Table 2. Uptake of protein and factor X from pig serum by various forms of calcium phosphate Details as in Table 1 , except that the protein was eluted with 0.25~-potassiumphosphate buffer, pH7.0, a adsorbents. Hydroxyapatite was prepared by the method of Tiselius et al. (1956). Calcium phosphate gel Poole, Dorset, U.K. Adsorbent Hydroxyapatite Calcium phosphate gel Calcium orthophosphate
Protein eluted (pglrng of adsorbent) 99 298 37
Total factor X activity (units/g of adsorbent) 4.7 8.5 0.8
lo2 x Sp (units/m
720
BIOCHEMICAL SOCIETY TRANSACTIONS
the pH range 2-6. From pH6 to 10.2 the mobilities of the protein-coated high-density particles accelerated to a level 35 % greater, at pH8.2, than the low-density particles. It is difficult at this stage to provide a complete explanation of this electrophoretic behaviour, but we consider it likely that on the smooth flat faces of the high-density particles, the protein is adsorbed as an expanded monolayer and allows a higher particle mobility from pH6 to 10.2, owing to the exposure of charged side groups (Chattoraj & Bull, 1959). In contrast, the uptake of adsorbed protein within the porous aggregates of low-density BaSOI (Howell & Deacon, 1975) prevents the increased mobility owing to unfolding of the protein. If this interpretation is valid, it would support the proposal of Howell & Dupe (1972) that partial unfolding on this adsorbent encourages autoproteolysis of factor X leading to its activation. For protein adsorbed to particles of barium citrate the pH-mobility curve showed a slight displacement in the acid region compared with the bare particles and reflects the small amount of protein adsorbed. The pH-mobility curve obtained for barium citrate precipitated in situ was interesting in that the linear variation of mobility with pH is typical of non-ionogenic particles (Goddard, 1970). This supports the proposal of Tishkoff et al. (1968) that the protein is sequestered within a crystal lattice, and in this state, it is unlikely that any unfolding and activation can occur. S. L. M. D. acknowledges receipt of a Science Research Council studentship.
Chattoraj, D. K. & Bull, H. B. (1959) J. Am. Chem. SOC.81, 5128-5136 Goddard, G. H. (1970) Ph.D. Thesis, University of London Howell, R. M. & Deacon, S. L. M. (1975) Thromb. Diarh. Haemorrh. 33, 256-270 Howell, R. M. & Dupe, R. J. (1972) Thromb. Diath.Haemorrh. 28, 306-316 Howell, R. M. & Scott, G . B. D. (1964) Br. J. Exp. Pathol. 45, 618-626 Lowry,O. H., Rosebrough,N.J., Farr, A. L. &Randall, R. J. (1951)J.Biol.Chem. 193,265-275 Stenflo, J., Fernlund, P., Egan, W. & Roepstorff, P. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 273C-2733 Tiselius, A., HjCrten, S. 0. & Levin, 6.(1956) Arch. Biochem. Biophys. 65, 132-155 Tishkoff,G . H., Williams, L. C. & Brown, D. M. (1968) J. Biol. Chem. 243,4151-4167
Physical Studies on Calcium Phosphate and Barium Citrate Used as Clotting-Factor Adsorbents SUSAN L. M. DEACON and ROY M. HOWELL Department of Biochemistry, Queen Elizabeth College, London W8 7AH, U.K.
Inorganic precipitates, containing Ca2+or BaZ+cations, have been used extensively in purification procedures for removing the vitamin K-dependent clotting factors from serum or plasma. The prothrombin group, which consists of factors 11, VII, IX and X, contains the modified amino acid y-carboxyglutamic acid which is responsible for the high cation-binding capacity of these proteins (Stenflo et al., 1974). We have examined the crystalline structure and charge of these inorganic compounds more closely, by scanning electron microscopy and particle electrophoresis, in a n attempt to relate physical properties and chemical composition to their properties as clotting-factor adsorbents. The present investigation on the physical properties of calciumphosphate and bariumcitrate is similar to our previous study of BaS04asaclottingfactor adsorbent (Howell & Deacon, 1975). The electrophoretic mobility of particles of various adsorbents was measured at 25°C in 0.02~-barbitonebuffer over the pH range 2-1 1 by the procedure of Bangham et al. (1958). Despite differences in morphology, various grades of BaSO,, in the former study, gave similar pH-mobility curves, since the internal composition of the crystal remains relatively constant. Incontrast, the present study showed a considerable shift in theshape 1976
