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Adsorption of Succinylated Lysozyme on Hydroxyapatite
JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.
189, 37–42 (1997)
CS974787
Adsorption of Succinylated Lysozyme on Hydroxyapatite ALLAL BARROUG,* ,1 JACQUES FASTREZ,† JACQUES LEMAITRE,‡
AND
PAUL ROUXHET§
*Laboratoire de Chimie Physique, Faculte´ des Sciences Semlalia, Universite´ Cadi Ayyad, B.P. S.15, 40001 Marrakech, Morocco; †De´partement de Biologie, Faculte´ des Sciences, Universite´ Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium; ‡Laboratoire de Technologie des Poudres, Ecole Polytechnique Fe´de´rale de Lausanne, CH-1015 Lausanne, Switzerland; and §Unite´ de Chimie des Interfaces, Faculte´ des Sciences Agronomiques, Croix du sud 2/18, B-1348 Louvain-la-Neuve, Belgium Received July 15, 1996; accepted January 21, 1997
tories. Its chemical and textural characteristics have been reported before under the name HAP III (2); its surface area is 80 m2rg 01 . Hen egg white lysozyme (Sigma No. 6876, Grade 1) was succinylated following the method of Bernard et al. (3). A large excess of succinic anhydride (mole fraction with respect to the protein is about 360) was used in order to achieve a high degree of substitution. At room temperature (22 { 2) 7C, 2 g of lysozyme were dissolved in 100 cm/3 of a 50 mmolrdm03 hepes buffer at pH 8.2 (Janssen Chimica) and NaCl was added to reach 1.25 molrdm03 . After dissolution, 5 g of anhydrous succinic (Janssen Chimica) dissolved in 5 cm/3 of dioxane was added slowly, while the pH was maintained at 8.2 by addition of 2 molrdm03 NaOH. The mixture was stirred for 2 h at room temperature. In order to regenerate the hydroxyl functions which could have reacted, the solution was mixed with the same volume of 0.5 molrdm03 buffer carbonate at pH 10 and the mixture was stirred for 20 h at 377C (3, 4). The solution of succinylated lysozyme was dialyzed for 4 days against 1 to 2 dm/3 of demineralized water, the latter being replaced three times. It was then freeze-dried (Edwards freeze dryer, condenser temperature 213 K, residual pressure 200–300 Pa). The remaining amino functions of the modified lysozyme were determined by 2, 4, 6-trinitrobenzenesulfonic acid (4, 5). The degree of succinylation was found to be about 96%, assuming that seven amine functions per molecule are able to react. The purity of the modified protein was characterized by fast protein liquid chromatography (Pharmacia Fine Chemicals) with an anion exchange column (Pharmacia Fine Chemicals, mono Q HR515). The latter was equilibrated with a solution of 20 mmolrdm03 distilled ethanolamine (pH 9). The protein solution (0.1 cm/3 , 1 mg proteinrcm03 ) was then injected and elution was carried out by increasing the ionic strength (ethanolamine 20 mmolrdm03 , NaCl from 0 to 0.5 molrdm03 , pH 9). The flow rate was 1 cm/3rmin 01 . The chromatogram obtained is presented in Fig. 1. All the
Hen egg white lysozyme has been succinylated, the isoelectric point being shifted thereby from about 11 to 4.5–4.7. Its adsorption on a chromatography grade hydroxyapatite has been investigated at 207C and in the pH range from 5.9 to 7.4. Adsorption takes place despite an electrical repulsion between the surface and the adsorbate; consequently, it is favored by a decrease of pH and phosphate concentration and an increase of calcium concentration and ionic strength. While adsorption of native lysozyme is driven by electrostatic attraction to the surface, adsorption of succinylated lysozyme is controlled by hydrophobic interactions. This may be attributed to a different structure of modified lysozyme, compared to native lysozyme, or to a greater tendency to undergo conformational changes. q 1997 Academic Press Key Words: hydroxyapatite; adsorption; lysozyme adsorption; succinylated lysozyme; zeta potential; hydrophobic interactions.
INTRODUCTION
The adsorption of hen egg white lysozyme (isoelectric point near pH 11) by various apatites has been investigated (1, 2). The adsorption process is fast and reversible with respect to dilution. It is governed by protein surface electrostatic attraction and consists in a surface neutralization, as demonstrated by electrophoretic mobility measurements and by the influence of the liquid composition (pH, ionic strength, concentration of potential determining calcium and phosphate ions). This paper reports on the adsorption of hen egg white lysozyme, chemically modified to decrease the isoelectric point in such a way that the protein be negatively charged in the pH range used. Therefore the amine groups were derivatized by the succinyl groups. MATERIALS
The adsorbent is a hydroxyapatite used for liquid phase chromatography, Bio-gel HTP, supplied by BIO-Rad labora1
To whom correspondence should be addressed.
37
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0021-9797/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BARROUG ET AL.
FIG. 1. FPLC chromatogram of succinylated lysozyme on an anion exchange resin. The ionic strength variation used for elution is indicated by the interupted line.
grade). The latter was introduced in the form of a solution of KH2PO4 and K2HPO4 , 0.1 molrdm03 of each, the pH of which was 6.8. In certain cases, the pH was modified by HNO3 or KOH. The protein was dissolved in these solutions. The adsorbent (15 mg, unless otherwise stated) was dispersed in 1.36 cm/3 of the electrolyte solution in a polypropylene tube, sonicated for 3 to 5 min, and stirred for 24 h. A portion (0.14 cm/3 ) of the modified lysozyme solution was introduced into the adsorbent suspension; the protein concentration covered the range 0.05–2 grdm03 . Unless otherwise stated, the mixture was stirred for 8 to 10 h at room temperature (20 { 2) 7C and then centrifuged for 10 min at 8000 g. The protein concentration in solution was determined by measuring the absorbance at 282 nm with a Beckmann ACTA M IV Spectrometer. The experimental determination of the absorption coefficient made by considering the modified lysozyme sample as 100% pure gave a value of (33.0 { 0.8) 10 6 mol 01rcm2 . The pH of the solution was measured before and after adsorption. Blanks performed without apatite showed that succinylated lysozyme was completely recovered and, consequently, did not adsorb on propylene to any measurable extent. Desorption was examined by replacing the supernatant with the same volume of a solution equilibrated with the pure adsorbent. After 18 h of stirring, the succinylated lysozyme concentration was determined in the solution. Electrophoretic Mobility Measurements
peaks found correspond to fractions eluted in a range of ionic strengths of 0.3 to 0.4, indicating that they have similar charge properties. The most abundant fraction represents about 45% of the product. Note that the native lysozyme sample containts 15% of contaminants, the charge of which is very close to that of lysozyme. The presence of various components in the solution of succinylated lysozyme is attributed to unreacted lysine residues or to the existence of various conformations. The isoelectric point of the modified protein was determined by isoelectric focusing (Pharmacia FBE 3000) on agarose gel (IEF Pharmacia), following procedures described by Pharmacia (6). The calibration kit (Low PI Kit, Pharmacia) covered the range from pH 2.5 to 6.5. An intense and narrow spot at pH 4.70 { 0.20 was accompanied by a less intense spot at pH 4.55 { 0.20. The observed isoelectric point is in agreement with the data of Kooistra et al. (7). For the calculations below, the succinylated lysozyme was assigned a molecular weight of about 15,300. METHODS
Adsorption Measurements Solutions were prepared with a given concentration of KNO3 and Ca(NO3 )2 or phosphate (Merck, analytical
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The electrophoretic mobility of the adsorbent was determined with a Pen Kem Lazer-Zee Meter, model 500, with a polymethyl methacrylate cell. The solutions containing 1 mmolrdm03 KNO3 were adjusted to the desired pH by 0.1 molrdm03 HNO3 or KOH solutions. The suspensions were prepared by introducing 20 mg of solid in 100 cm/3 of the solution. They were placed in a polyethylene flask and sonicated for 3 to 5 min. They were stirred at room temperature (20 { 2) 7C for 24 h with a vertical support rotating at 40 rpm. The solution was then in equilibrium with the solid. In order to measure the electrophoretic mobility after adsorption of succinytated lysozyme, the latter was added (65 mmolrdm03 ) to the suspensions described above and the mixture was incubated overnight. RESULTS
The kinetics of succinylated lysozyme adsorption was examined in 3 mmolrdm03 KNO3 (pH 7.80), 1 mmolrdm03 KNO3 / 1 mmolrdm03 Ca(NO3 )2 (pH 5.95), and 2 mmolrdm03 phosphate (pH 6.60); the ionic strength was 4.5 mmolrdm03 in all cases. The adsorbed amount no longer changed after 2–3 h.
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ADSORPTION OF LYSOZYME ON HYDROXYAPATITE
to the isoelectric point of the protein, the amount of protein adsorbed and the apparent affinity constant increase. An increase of the ionic strength (part C) leads to a higher value of the maximum amount adsorbed but to a decrease of K. Electrophoretic mobility curves versus pH, determined in 1 mmolrdm03 KNO3 in the absence and in the presence of the protein, are presented in Fig. 3. The pH range examined in the adsorption experiments lies above the isoelectric point. The electrophoretic mobility and thus the electrophoretic potential are found to be more negative after adsorption of succinylated lysozyme. It may be noted that the adsorption improves the dispersion of hydroxyapatite suspension, which remains turbid after sedimentation for a few hours. DISCUSSION
Succinylated lysozyme adsorbs on hydroxyapatite under conditions such that both the adsorbate and the adsorbent are negatively charged. Similar observations were made for
FIG. 2. Adsorption ( j ) and desorption ( h ) isotherms of succinylated lysozyme on HAP III (10 grdm03 ) in 1 mmolrdm03 KNO3 solutions at pH 6.8.
Figure 2 presents the adsorption and desorption isotherms in 1 mmolrdm03 KNO3 solution, at pH 6.3. The protein is desorbed upon dilution. However, the desorption and adsorption branches are not superimposed, indicating that the process is not totally reversible. The adsorption isotherms obtained with different solution compositions are well described by the Langmuir equation. They have been linearized by plotting the amount adsorbed per unit area (G) versus G/C, C being the stationary concentration in the solution. This provided a normalized way to determine the maximum amount adsorbed, N. The slop provided also an apparent affinity constant, K. This is only apparent as the process is not perfectly reversible. However, it makes it possible to examine the influence of experimental conditions on the sharpness of the increase of the amount adsorbed versus concentration at low coverage. N and K have been determined by linear regression and are given in Table 1 for various solution compositions. A comparison (part A) of the adsorption parameters in KNO3 and phosphate solutions at the same pH (6.5–6.7) and ionic strength (4.5 mmolrdm03 ) shows that the presence of phosphate excess in the solution is unfavorable to the adsorption of succinylated lysozyme. On the other hand, an excess of calcium is favorable to adsorption. The influence of pH has been examined in 1 mmolrdm03 KNO3 , in the range from 5.90 to 7.40 (part B). As pH decreases and becomes closer
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TABLE 1 Influence of the Solution Composition on the Adsorption Parameters of Succinylated Lysozyme on HAP III: Maximum Adsorbed Amount (N) and Apparent Affinity Constant (K) { 95% Confidence Interval A. Influence of the added electrolyte: ionic strength 4.5 mmolrdm03 Electrolyte added
Final pH ({0.05)
N { IC (nmolrm02)
K { IC (mmol01rdm3)
6.0 6.5
73.0 { 9.0 42.7 { 6.8
1065 { 440 78.4 { 28.6
6.7
11.8 { 7.7
Ca(NO3)2 1 mmolrdm03 / KNO3 1 mmolrdm03 KNO3 1 mmolrdm03 Phosphate 2 mmolrdm03
28
{ 30
B. Influence of pH: 1 mmolrdm03 KNO3 Final pH ({0.05)
N { IC (nmolrm02)
K { IC (mmol01rdm3)
5.95 6.85 7.40
81.9 { 5.2 36.0 { 2.7 18.0 { 15.2
366.0 { 75.0 125.4 { 27.4 25.7 { 40.6
C. Influence of ionic strength (I): addition of KNO3 I total (mmolrdm03)
Final pH ({0.05)
2 4.5 10 25 46
6.85 6.50 6.40 6.75 6.65
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N { IC (nmolrm02) 36.0 54.7 45.1 57.0 79.3
{ { { { {
2.7 6.8 5.8 3.5 41.0
K { IC (mmol01rdm3) 125.0 78.4 75.4 59.2 15.0
{ { { { {
27.0 28.6 22.4 7.4 10.8
40
BARROUG ET AL.
repulsion for the former and attraction for the latter. According to data reported for different proteins and adsorbents (15, 17, 18), the adsorption of succinylated lysozyme would be due to hydrophobic interactions. It would be driven by entropy and might be related to structural rearrangements in the protein molecule. The amount of succinylated lysozyme adsorbed at the maximum coverage increases with the ionic strength. This is due to improved screening of the electrical charges. The concomitant decrease of K must be considered with caution as the adsorption process is not strictly reversible and the adsorbate is not a pure substance. In the case of the adsorption of human plasma albumin on polystyrene latex, both being negatively charged, the apparent affinity at low cover-
FIG. 3. Electrophoretic mobility versus pH curve of HAP III dispersed (0.2 grdm03 ) in 1 mmolrdm03 KNO3 ( h ) and in the same solution containing 65 mmolrdm03 of succinylated lysozyme ( j ).
the adsorption of human serum plasma albumin (8), bovine serum albumin (9), and numerous acidic proteins (10, 11) on hydroxyapatite and for the adsorption of albumin on various adsorbents (12, 13). This is opposed to the observations made for native lysozyme (2), the adsorption of which is driven by adsorbent–adsorbate electrostatic attraction. The electrokinetic measurements provide a direct confirmation that adsorption involves negatively charged adsorbate and adsorbent. As a matter of fact, adsorption of succinylated lysozyme makes the electrokinetic potential of hydroxyapatite more negative and, accordingly, favors its dispersion. The adsorption of negatively charged bovine serum albumin molecules induces a negative surface charge on hydroxyapatite at pH 6.0 (9). This is indeed in opposition to the adsorption of native lysozyme, which involves neutralization of the surface charge by the adsorbate. The native and succinylated lysozyme also differ from each other in adsorption kinetics. For the former, adsorption equilibrium is reached within minutes, while for the latter, a stationary state is obtained after 2–3 h of incubation, indicating that electrostatic repulsion causes reduced adsorption rates. The slowness of this process may be attributed to a potential barrier which the protein molecules have to overcome to adsorb and/or to a conformational change of the protein (14–16). Figure 4 shows that the influence of pH on the adsorption isotherms of succinylated lysozyme is opposite to that for native lysozyme. The influence of the potential determining ions is also consistent with an adsorbate–adsorbent electrical
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FIG. 4. Adsorption of succinylated ( h ) and native lysozyme ( j ) on HAP III in 1 mmolrdm03 KNO3 solution: maximum amount of protein adsorbed (N) and apparent affinity constant (K) vs pH.
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ADSORPTION OF LYSOZYME ON HYDROXYAPATITE
age was also found to decrease when the ionic strength increased (19). This was attributed to an increased conformational stability of the protein, limiting hydrophobic interactions and/or to co-adsorption of ions. The same explanation may be proposed for the succinylated lysozyme–hydroxyapatite system. In the presence of a 1 mmolrdm03 KNO3 solution at pH 5.95, the maximum adsorbed amounts are 13.7 and 91.9 nmolrm02 for native (2) and succinylated lysozyme, respectively. The apparent affinity constants are 13 and 366 mmol 01rdm3 . Under these conditions, the surface is weakly negatively charged. Besides, a small amount of native lysozyme is adsorbed through charge neutralization. In spite of electrical repulsion with the surface, adsorption of modified lysozyme takes place extensively due to hydrophobic interactions. An overall comparison of the adsorption of native and succinylated lysozyme under different conditions shows that the isotherm parameters fall in the same range; the maximum adsorbed amount varies from 12 to 82 nmolrm02 and the apparent affinity constant varies from 0.01 to 1 mmol 01rdm3 . This similarity is surprising as the adsorption mechanisms are completely different and the solution characteristics (pH and phosphate and calcium concentration) influence adsorption in opposite directions. While succinylation of lysozyme cancels the possibility of an electrostatic attraction with hydroxyapatite under the conditions investigated, it confers thus to the protein a stronger tendency to form hydrophobic interactions (15, 16). Considering a molecular diameter of 3.25 nm (20) and assuming that the charged moieties are located on the molecular periphery, the surface density of charge would be /0.21 nm02 for native lysozyme in the pH range from 6 to 8 (eight positive charges per molecule) and 00.16 nm02 for modified lysozyme (six succinyl radicals per molecule). Consequently, succinylation as such should not appreciably modify the hydrophobicity if lysozyme molecule remains compact. The ability of the modified protein to adsorb via hydrophobic interactions may be attributed to a different structure or a greater tendency to undergo conformational changes, compared to the native form. It has been reported that acetylation of lysozyme and of ribonuclease prevents retention of these proteins by hydroxyapatite (10). This may be due to a decrease of the electrostatic adsorbent–adsorbate attraction without compensation by an increased tendency to form hydrophobic interactions. The adsorption of succinylated lysozyme on hydroxyapatite takes place under conditions of electrostatic repultions as shown by the solution characteristics as well as electrophoretic mobility measurements. The driving forces of the adsorption process seem to be structural rearrangements in the protein molecule. This has been demonstrated, from experimental data, by a decrease in
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the secondary structure ( a-helix and b-sheet ) in the adsorbed protein ( 21 ) . The results obtained for catalase adsorbed on hydroxyapatite, under conditions where both are negatively charged, illustrate the conformational change in the adsorbed protein ( 22 ) ; the enzyme adsorbed is about four times less active than the free enzyme in solution. This may be explained by the unfolding of the enzyme on solid surface and would indicate a departure from the active conformation. Similar observations were made for the same protein adsorbed on carbon black ( 23 ) and for b-D-glucosidase adsorbed on mineral surfaces ( 24 ) . CONCLUSION
Although the product of lysozyme succinylation was not a pure substance, it is clear that it adsorbs on hydroxyapatite in spite of electrostatic repulsion of the surface. This has been demonstrated by the influence of pH, ionic strength, and concentration of potential determining ions as well as direct electrophoretic mobility measurements. Succinylation produces more than a change of the isoelectric point of lysozyme. The ability of the protein to form hydrophobic interactions is increased because of either a structural change or a stronger tendency to undergo conformational changes. Thereby, hydrophobic interactions overpass adsorbate–adsorbent electrostatic repulsions. ACKNOWLEDGMENTS The support of the Service de Programmation de la Politique Scientifique (Concerted Action Physical Chemistry of Interfaces and Biotechnology) and the Research Associates of the FNRS (Belgium) is gratefully acknowledged.
REFERENCES 1. Barroug, A., Ph.D. Thesis, Universite´ Catholique de Louvain, 1989. 2. Barroug, A., Lemaitre, J., and Rouxhet, P. G., Colloids Surf. 37, 339 (1989). 3. Bernad, A., Nieto, M. A., Vioque, A., and Palacian, E., Biochim. Biophys. Acta 873, 350 (1986). 4. Nieto, M. A., and Palacian, E., Biochim. Biophys. Acta 749, 204 (1983). 5. Habeeb, A. F. S. A., Arch. Biochim. Biophys. 121, 652 (1967). 6. Pharmacia Fine Chemicals, ‘‘Isoelectric focusing—Principles and methods,’’ Uppsala, Sweden, 1982. 7. Kooistra, T., Duursma, A. M., Bouma, J. M. W., and Gruber, M., Biochim. Biophys. Acta 631, 439 (1980). 8. Hlady, V., and Fu¨redi-Milhofer, H., J. Colloid Interface Sci. 69, 460 (1979). 9. Kandori, K., Sawai, S., Yamamato, Y., Saito, H., and Ishikawa, T., Colloids Surf. 68, 283 (1992). 10. Gorbunoff, M. J., Anal. Biochem. 136, 425 (1984). 11. Kawasaki, T., J. Chromatogr. 544, 147 (1991). 12. Norde, W., and Lyklema, J., Colloids Surf. 38, 1 (1989).
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13. Elgersma, A. V., Zsom, R. L. J., Norde, W., and Lyklema, J., J. Colloids Surf. 54, 89 (1991). 14. Van Dulm, P., and Norde, W., J. Colloid Interface Sci. 91, 248 (1983). 15. Norde, W., Adv. Colloid Interface Sci. 25, 267 (1986). 16. Norde, W., and Lyklema, J., J. Biomater. Sci. Polym. Edu. 3, 183 (1991). 17. Koutsoukos, P. G., Norde, W., and Lyklema, J., J. Colloid Interface Sci. 95, 385 (1983). 18. Norde, W., Arai, T., and Shirahama, H., Biofouling 4, 37 (1991).
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19. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 20. Imoto, T., Johnson, L. N., North, A. C. T., Phillips, R. C., and Rupley, J. A., Enzymes 7, 665 (1972). 21. Arai, T., and Norde, W., Colloids Surf. 51, 1 (1990). 22. Barroug, A., Lernoux, E., Lemaitre, J., and Rouxhet, P. G., to be submitted for publication. 23. Sosa, R. C., Masy, D., and Rouxhet, P. G., Carbon 32, 1369 (1994). 24. Quiquampoix, H., Chassin, C., and Ratcliffe, G., Prog. Colloid Polym. Sci. 79, 59 (1989).
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CS974787
Adsorption of Succinylated Lysozyme on Hydroxyapatite ALLAL BARROUG,* ,1 JACQUES FASTREZ,† JACQUES LEMAITRE,‡
AND
PAUL ROUXHET§
*Laboratoire de Chimie Physique, Faculte´ des Sciences Semlalia, Universite´ Cadi Ayyad, B.P. S.15, 40001 Marrakech, Morocco; †De´partement de Biologie, Faculte´ des Sciences, Universite´ Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium; ‡Laboratoire de Technologie des Poudres, Ecole Polytechnique Fe´de´rale de Lausanne, CH-1015 Lausanne, Switzerland; and §Unite´ de Chimie des Interfaces, Faculte´ des Sciences Agronomiques, Croix du sud 2/18, B-1348 Louvain-la-Neuve, Belgium Received July 15, 1996; accepted January 21, 1997
tories. Its chemical and textural characteristics have been reported before under the name HAP III (2); its surface area is 80 m2rg 01 . Hen egg white lysozyme (Sigma No. 6876, Grade 1) was succinylated following the method of Bernard et al. (3). A large excess of succinic anhydride (mole fraction with respect to the protein is about 360) was used in order to achieve a high degree of substitution. At room temperature (22 { 2) 7C, 2 g of lysozyme were dissolved in 100 cm/3 of a 50 mmolrdm03 hepes buffer at pH 8.2 (Janssen Chimica) and NaCl was added to reach 1.25 molrdm03 . After dissolution, 5 g of anhydrous succinic (Janssen Chimica) dissolved in 5 cm/3 of dioxane was added slowly, while the pH was maintained at 8.2 by addition of 2 molrdm03 NaOH. The mixture was stirred for 2 h at room temperature. In order to regenerate the hydroxyl functions which could have reacted, the solution was mixed with the same volume of 0.5 molrdm03 buffer carbonate at pH 10 and the mixture was stirred for 20 h at 377C (3, 4). The solution of succinylated lysozyme was dialyzed for 4 days against 1 to 2 dm/3 of demineralized water, the latter being replaced three times. It was then freeze-dried (Edwards freeze dryer, condenser temperature 213 K, residual pressure 200–300 Pa). The remaining amino functions of the modified lysozyme were determined by 2, 4, 6-trinitrobenzenesulfonic acid (4, 5). The degree of succinylation was found to be about 96%, assuming that seven amine functions per molecule are able to react. The purity of the modified protein was characterized by fast protein liquid chromatography (Pharmacia Fine Chemicals) with an anion exchange column (Pharmacia Fine Chemicals, mono Q HR515). The latter was equilibrated with a solution of 20 mmolrdm03 distilled ethanolamine (pH 9). The protein solution (0.1 cm/3 , 1 mg proteinrcm03 ) was then injected and elution was carried out by increasing the ionic strength (ethanolamine 20 mmolrdm03 , NaCl from 0 to 0.5 molrdm03 , pH 9). The flow rate was 1 cm/3rmin 01 . The chromatogram obtained is presented in Fig. 1. All the
Hen egg white lysozyme has been succinylated, the isoelectric point being shifted thereby from about 11 to 4.5–4.7. Its adsorption on a chromatography grade hydroxyapatite has been investigated at 207C and in the pH range from 5.9 to 7.4. Adsorption takes place despite an electrical repulsion between the surface and the adsorbate; consequently, it is favored by a decrease of pH and phosphate concentration and an increase of calcium concentration and ionic strength. While adsorption of native lysozyme is driven by electrostatic attraction to the surface, adsorption of succinylated lysozyme is controlled by hydrophobic interactions. This may be attributed to a different structure of modified lysozyme, compared to native lysozyme, or to a greater tendency to undergo conformational changes. q 1997 Academic Press Key Words: hydroxyapatite; adsorption; lysozyme adsorption; succinylated lysozyme; zeta potential; hydrophobic interactions.
INTRODUCTION
The adsorption of hen egg white lysozyme (isoelectric point near pH 11) by various apatites has been investigated (1, 2). The adsorption process is fast and reversible with respect to dilution. It is governed by protein surface electrostatic attraction and consists in a surface neutralization, as demonstrated by electrophoretic mobility measurements and by the influence of the liquid composition (pH, ionic strength, concentration of potential determining calcium and phosphate ions). This paper reports on the adsorption of hen egg white lysozyme, chemically modified to decrease the isoelectric point in such a way that the protein be negatively charged in the pH range used. Therefore the amine groups were derivatized by the succinyl groups. MATERIALS
The adsorbent is a hydroxyapatite used for liquid phase chromatography, Bio-gel HTP, supplied by BIO-Rad labora1
To whom correspondence should be addressed.
37
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JCIS 4787
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04-07-97 20:49:51
0021-9797/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BARROUG ET AL.
FIG. 1. FPLC chromatogram of succinylated lysozyme on an anion exchange resin. The ionic strength variation used for elution is indicated by the interupted line.
grade). The latter was introduced in the form of a solution of KH2PO4 and K2HPO4 , 0.1 molrdm03 of each, the pH of which was 6.8. In certain cases, the pH was modified by HNO3 or KOH. The protein was dissolved in these solutions. The adsorbent (15 mg, unless otherwise stated) was dispersed in 1.36 cm/3 of the electrolyte solution in a polypropylene tube, sonicated for 3 to 5 min, and stirred for 24 h. A portion (0.14 cm/3 ) of the modified lysozyme solution was introduced into the adsorbent suspension; the protein concentration covered the range 0.05–2 grdm03 . Unless otherwise stated, the mixture was stirred for 8 to 10 h at room temperature (20 { 2) 7C and then centrifuged for 10 min at 8000 g. The protein concentration in solution was determined by measuring the absorbance at 282 nm with a Beckmann ACTA M IV Spectrometer. The experimental determination of the absorption coefficient made by considering the modified lysozyme sample as 100% pure gave a value of (33.0 { 0.8) 10 6 mol 01rcm2 . The pH of the solution was measured before and after adsorption. Blanks performed without apatite showed that succinylated lysozyme was completely recovered and, consequently, did not adsorb on propylene to any measurable extent. Desorption was examined by replacing the supernatant with the same volume of a solution equilibrated with the pure adsorbent. After 18 h of stirring, the succinylated lysozyme concentration was determined in the solution. Electrophoretic Mobility Measurements
peaks found correspond to fractions eluted in a range of ionic strengths of 0.3 to 0.4, indicating that they have similar charge properties. The most abundant fraction represents about 45% of the product. Note that the native lysozyme sample containts 15% of contaminants, the charge of which is very close to that of lysozyme. The presence of various components in the solution of succinylated lysozyme is attributed to unreacted lysine residues or to the existence of various conformations. The isoelectric point of the modified protein was determined by isoelectric focusing (Pharmacia FBE 3000) on agarose gel (IEF Pharmacia), following procedures described by Pharmacia (6). The calibration kit (Low PI Kit, Pharmacia) covered the range from pH 2.5 to 6.5. An intense and narrow spot at pH 4.70 { 0.20 was accompanied by a less intense spot at pH 4.55 { 0.20. The observed isoelectric point is in agreement with the data of Kooistra et al. (7). For the calculations below, the succinylated lysozyme was assigned a molecular weight of about 15,300. METHODS
Adsorption Measurements Solutions were prepared with a given concentration of KNO3 and Ca(NO3 )2 or phosphate (Merck, analytical
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The electrophoretic mobility of the adsorbent was determined with a Pen Kem Lazer-Zee Meter, model 500, with a polymethyl methacrylate cell. The solutions containing 1 mmolrdm03 KNO3 were adjusted to the desired pH by 0.1 molrdm03 HNO3 or KOH solutions. The suspensions were prepared by introducing 20 mg of solid in 100 cm/3 of the solution. They were placed in a polyethylene flask and sonicated for 3 to 5 min. They were stirred at room temperature (20 { 2) 7C for 24 h with a vertical support rotating at 40 rpm. The solution was then in equilibrium with the solid. In order to measure the electrophoretic mobility after adsorption of succinytated lysozyme, the latter was added (65 mmolrdm03 ) to the suspensions described above and the mixture was incubated overnight. RESULTS
The kinetics of succinylated lysozyme adsorption was examined in 3 mmolrdm03 KNO3 (pH 7.80), 1 mmolrdm03 KNO3 / 1 mmolrdm03 Ca(NO3 )2 (pH 5.95), and 2 mmolrdm03 phosphate (pH 6.60); the ionic strength was 4.5 mmolrdm03 in all cases. The adsorbed amount no longer changed after 2–3 h.
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ADSORPTION OF LYSOZYME ON HYDROXYAPATITE
to the isoelectric point of the protein, the amount of protein adsorbed and the apparent affinity constant increase. An increase of the ionic strength (part C) leads to a higher value of the maximum amount adsorbed but to a decrease of K. Electrophoretic mobility curves versus pH, determined in 1 mmolrdm03 KNO3 in the absence and in the presence of the protein, are presented in Fig. 3. The pH range examined in the adsorption experiments lies above the isoelectric point. The electrophoretic mobility and thus the electrophoretic potential are found to be more negative after adsorption of succinylated lysozyme. It may be noted that the adsorption improves the dispersion of hydroxyapatite suspension, which remains turbid after sedimentation for a few hours. DISCUSSION
Succinylated lysozyme adsorbs on hydroxyapatite under conditions such that both the adsorbate and the adsorbent are negatively charged. Similar observations were made for
FIG. 2. Adsorption ( j ) and desorption ( h ) isotherms of succinylated lysozyme on HAP III (10 grdm03 ) in 1 mmolrdm03 KNO3 solutions at pH 6.8.
Figure 2 presents the adsorption and desorption isotherms in 1 mmolrdm03 KNO3 solution, at pH 6.3. The protein is desorbed upon dilution. However, the desorption and adsorption branches are not superimposed, indicating that the process is not totally reversible. The adsorption isotherms obtained with different solution compositions are well described by the Langmuir equation. They have been linearized by plotting the amount adsorbed per unit area (G) versus G/C, C being the stationary concentration in the solution. This provided a normalized way to determine the maximum amount adsorbed, N. The slop provided also an apparent affinity constant, K. This is only apparent as the process is not perfectly reversible. However, it makes it possible to examine the influence of experimental conditions on the sharpness of the increase of the amount adsorbed versus concentration at low coverage. N and K have been determined by linear regression and are given in Table 1 for various solution compositions. A comparison (part A) of the adsorption parameters in KNO3 and phosphate solutions at the same pH (6.5–6.7) and ionic strength (4.5 mmolrdm03 ) shows that the presence of phosphate excess in the solution is unfavorable to the adsorption of succinylated lysozyme. On the other hand, an excess of calcium is favorable to adsorption. The influence of pH has been examined in 1 mmolrdm03 KNO3 , in the range from 5.90 to 7.40 (part B). As pH decreases and becomes closer
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TABLE 1 Influence of the Solution Composition on the Adsorption Parameters of Succinylated Lysozyme on HAP III: Maximum Adsorbed Amount (N) and Apparent Affinity Constant (K) { 95% Confidence Interval A. Influence of the added electrolyte: ionic strength 4.5 mmolrdm03 Electrolyte added
Final pH ({0.05)
N { IC (nmolrm02)
K { IC (mmol01rdm3)
6.0 6.5
73.0 { 9.0 42.7 { 6.8
1065 { 440 78.4 { 28.6
6.7
11.8 { 7.7
Ca(NO3)2 1 mmolrdm03 / KNO3 1 mmolrdm03 KNO3 1 mmolrdm03 Phosphate 2 mmolrdm03
28
{ 30
B. Influence of pH: 1 mmolrdm03 KNO3 Final pH ({0.05)
N { IC (nmolrm02)
K { IC (mmol01rdm3)
5.95 6.85 7.40
81.9 { 5.2 36.0 { 2.7 18.0 { 15.2
366.0 { 75.0 125.4 { 27.4 25.7 { 40.6
C. Influence of ionic strength (I): addition of KNO3 I total (mmolrdm03)
Final pH ({0.05)
2 4.5 10 25 46
6.85 6.50 6.40 6.75 6.65
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N { IC (nmolrm02) 36.0 54.7 45.1 57.0 79.3
{ { { { {
2.7 6.8 5.8 3.5 41.0
K { IC (mmol01rdm3) 125.0 78.4 75.4 59.2 15.0
{ { { { {
27.0 28.6 22.4 7.4 10.8
40
BARROUG ET AL.
repulsion for the former and attraction for the latter. According to data reported for different proteins and adsorbents (15, 17, 18), the adsorption of succinylated lysozyme would be due to hydrophobic interactions. It would be driven by entropy and might be related to structural rearrangements in the protein molecule. The amount of succinylated lysozyme adsorbed at the maximum coverage increases with the ionic strength. This is due to improved screening of the electrical charges. The concomitant decrease of K must be considered with caution as the adsorption process is not strictly reversible and the adsorbate is not a pure substance. In the case of the adsorption of human plasma albumin on polystyrene latex, both being negatively charged, the apparent affinity at low cover-
FIG. 3. Electrophoretic mobility versus pH curve of HAP III dispersed (0.2 grdm03 ) in 1 mmolrdm03 KNO3 ( h ) and in the same solution containing 65 mmolrdm03 of succinylated lysozyme ( j ).
the adsorption of human serum plasma albumin (8), bovine serum albumin (9), and numerous acidic proteins (10, 11) on hydroxyapatite and for the adsorption of albumin on various adsorbents (12, 13). This is opposed to the observations made for native lysozyme (2), the adsorption of which is driven by adsorbent–adsorbate electrostatic attraction. The electrokinetic measurements provide a direct confirmation that adsorption involves negatively charged adsorbate and adsorbent. As a matter of fact, adsorption of succinylated lysozyme makes the electrokinetic potential of hydroxyapatite more negative and, accordingly, favors its dispersion. The adsorption of negatively charged bovine serum albumin molecules induces a negative surface charge on hydroxyapatite at pH 6.0 (9). This is indeed in opposition to the adsorption of native lysozyme, which involves neutralization of the surface charge by the adsorbate. The native and succinylated lysozyme also differ from each other in adsorption kinetics. For the former, adsorption equilibrium is reached within minutes, while for the latter, a stationary state is obtained after 2–3 h of incubation, indicating that electrostatic repulsion causes reduced adsorption rates. The slowness of this process may be attributed to a potential barrier which the protein molecules have to overcome to adsorb and/or to a conformational change of the protein (14–16). Figure 4 shows that the influence of pH on the adsorption isotherms of succinylated lysozyme is opposite to that for native lysozyme. The influence of the potential determining ions is also consistent with an adsorbate–adsorbent electrical
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FIG. 4. Adsorption of succinylated ( h ) and native lysozyme ( j ) on HAP III in 1 mmolrdm03 KNO3 solution: maximum amount of protein adsorbed (N) and apparent affinity constant (K) vs pH.
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ADSORPTION OF LYSOZYME ON HYDROXYAPATITE
age was also found to decrease when the ionic strength increased (19). This was attributed to an increased conformational stability of the protein, limiting hydrophobic interactions and/or to co-adsorption of ions. The same explanation may be proposed for the succinylated lysozyme–hydroxyapatite system. In the presence of a 1 mmolrdm03 KNO3 solution at pH 5.95, the maximum adsorbed amounts are 13.7 and 91.9 nmolrm02 for native (2) and succinylated lysozyme, respectively. The apparent affinity constants are 13 and 366 mmol 01rdm3 . Under these conditions, the surface is weakly negatively charged. Besides, a small amount of native lysozyme is adsorbed through charge neutralization. In spite of electrical repulsion with the surface, adsorption of modified lysozyme takes place extensively due to hydrophobic interactions. An overall comparison of the adsorption of native and succinylated lysozyme under different conditions shows that the isotherm parameters fall in the same range; the maximum adsorbed amount varies from 12 to 82 nmolrm02 and the apparent affinity constant varies from 0.01 to 1 mmol 01rdm3 . This similarity is surprising as the adsorption mechanisms are completely different and the solution characteristics (pH and phosphate and calcium concentration) influence adsorption in opposite directions. While succinylation of lysozyme cancels the possibility of an electrostatic attraction with hydroxyapatite under the conditions investigated, it confers thus to the protein a stronger tendency to form hydrophobic interactions (15, 16). Considering a molecular diameter of 3.25 nm (20) and assuming that the charged moieties are located on the molecular periphery, the surface density of charge would be /0.21 nm02 for native lysozyme in the pH range from 6 to 8 (eight positive charges per molecule) and 00.16 nm02 for modified lysozyme (six succinyl radicals per molecule). Consequently, succinylation as such should not appreciably modify the hydrophobicity if lysozyme molecule remains compact. The ability of the modified protein to adsorb via hydrophobic interactions may be attributed to a different structure or a greater tendency to undergo conformational changes, compared to the native form. It has been reported that acetylation of lysozyme and of ribonuclease prevents retention of these proteins by hydroxyapatite (10). This may be due to a decrease of the electrostatic adsorbent–adsorbate attraction without compensation by an increased tendency to form hydrophobic interactions. The adsorption of succinylated lysozyme on hydroxyapatite takes place under conditions of electrostatic repultions as shown by the solution characteristics as well as electrophoretic mobility measurements. The driving forces of the adsorption process seem to be structural rearrangements in the protein molecule. This has been demonstrated, from experimental data, by a decrease in
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41
the secondary structure ( a-helix and b-sheet ) in the adsorbed protein ( 21 ) . The results obtained for catalase adsorbed on hydroxyapatite, under conditions where both are negatively charged, illustrate the conformational change in the adsorbed protein ( 22 ) ; the enzyme adsorbed is about four times less active than the free enzyme in solution. This may be explained by the unfolding of the enzyme on solid surface and would indicate a departure from the active conformation. Similar observations were made for the same protein adsorbed on carbon black ( 23 ) and for b-D-glucosidase adsorbed on mineral surfaces ( 24 ) . CONCLUSION
Although the product of lysozyme succinylation was not a pure substance, it is clear that it adsorbs on hydroxyapatite in spite of electrostatic repulsion of the surface. This has been demonstrated by the influence of pH, ionic strength, and concentration of potential determining ions as well as direct electrophoretic mobility measurements. Succinylation produces more than a change of the isoelectric point of lysozyme. The ability of the protein to form hydrophobic interactions is increased because of either a structural change or a stronger tendency to undergo conformational changes. Thereby, hydrophobic interactions overpass adsorbate–adsorbent electrostatic repulsions. ACKNOWLEDGMENTS The support of the Service de Programmation de la Politique Scientifique (Concerted Action Physical Chemistry of Interfaces and Biotechnology) and the Research Associates of the FNRS (Belgium) is gratefully acknowledged.
REFERENCES 1. Barroug, A., Ph.D. Thesis, Universite´ Catholique de Louvain, 1989. 2. Barroug, A., Lemaitre, J., and Rouxhet, P. G., Colloids Surf. 37, 339 (1989). 3. Bernad, A., Nieto, M. A., Vioque, A., and Palacian, E., Biochim. Biophys. Acta 873, 350 (1986). 4. Nieto, M. A., and Palacian, E., Biochim. Biophys. Acta 749, 204 (1983). 5. Habeeb, A. F. S. A., Arch. Biochim. Biophys. 121, 652 (1967). 6. Pharmacia Fine Chemicals, ‘‘Isoelectric focusing—Principles and methods,’’ Uppsala, Sweden, 1982. 7. Kooistra, T., Duursma, A. M., Bouma, J. M. W., and Gruber, M., Biochim. Biophys. Acta 631, 439 (1980). 8. Hlady, V., and Fu¨redi-Milhofer, H., J. Colloid Interface Sci. 69, 460 (1979). 9. Kandori, K., Sawai, S., Yamamato, Y., Saito, H., and Ishikawa, T., Colloids Surf. 68, 283 (1992). 10. Gorbunoff, M. J., Anal. Biochem. 136, 425 (1984). 11. Kawasaki, T., J. Chromatogr. 544, 147 (1991). 12. Norde, W., and Lyklema, J., Colloids Surf. 38, 1 (1989).
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13. Elgersma, A. V., Zsom, R. L. J., Norde, W., and Lyklema, J., J. Colloids Surf. 54, 89 (1991). 14. Van Dulm, P., and Norde, W., J. Colloid Interface Sci. 91, 248 (1983). 15. Norde, W., Adv. Colloid Interface Sci. 25, 267 (1986). 16. Norde, W., and Lyklema, J., J. Biomater. Sci. Polym. Edu. 3, 183 (1991). 17. Koutsoukos, P. G., Norde, W., and Lyklema, J., J. Colloid Interface Sci. 95, 385 (1983). 18. Norde, W., Arai, T., and Shirahama, H., Biofouling 4, 37 (1991).
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19. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 20. Imoto, T., Johnson, L. N., North, A. C. T., Phillips, R. C., and Rupley, J. A., Enzymes 7, 665 (1972). 21. Arai, T., and Norde, W., Colloids Surf. 51, 1 (1990). 22. Barroug, A., Lernoux, E., Lemaitre, J., and Rouxhet, P. G., to be submitted for publication. 23. Sosa, R. C., Masy, D., and Rouxhet, P. G., Carbon 32, 1369 (1994). 24. Quiquampoix, H., Chassin, C., and Ratcliffe, G., Prog. Colloid Polym. Sci. 79, 59 (1989).
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