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Competitive and Cooperative Adsorptions of Bovine Serum Albumin and Lysozyme to Synthetic Calcium Hydroxyapatites Kazuhiko Kandori,* Megumi Mukai, Akemi Yasukawa, and Tatsuo Ishikawa School of Chemistry, Osaka University of Education, Asahigaoka 4-698-1, Kashiwara-shi, Osaka 582-8582, Japan Received May 26, 1999. In Final Form: October 29, 1999 Competitive and cooperative adsorptions of bovine serum albumin (BSA) and lysozyme (LSZ) on synthetic calcium hydroxyapatite (CaHap) were investigated at 15 °C in deionized distilled water of pH 6 by using a liquid chromatography method. The adsorption rate of LSZ was faster than that of BSA due to its larger diffusion coefficient, though the saturated adsorption amount of LSZ was less than that of BSA. The rates of BSA adsorption onto CaHap did not change by the addition of LSZ, while those of LSZ were considerably reduced by addition of BSA with forming (BSA--LSZ+) agglomerates in the solution. The cooperative adsorption behavior of LSZ was observed in the presence of lower amounts of BSA due to the preferential adsorption of the larger (BSA--LSZ+) agglomerates. However, in the case of higher BSA content, the adsorption of LSZ was inhibited by capturing the LSZ molecules in the (BSA--LSZ+) agglomerates. The similar cooperative adsorption behavior of BSA was also observed on all systems examined in the presence of various amounts of LSZ.
Introduction It is well-known that calcium hydroxyapatite [Ca10(PO4)6(OH)2, CaHap] has a high affinity to biomolecules with a large adsorption capacity and is nowadays widely used as a liquid chromatography adsorbent for separating biomolecules such as proteins.1-5 CaHap is also an excellent biomaterial from the viewpoint of biocompatibility. Since the CaHap surface is rapidly covered by proteineous layers in the body fluid when CaHap is implanted in body,6,7 the high adsorption capacity to biomolecules is the first requisite of CaHap for applying it in these biochemical fields because serum-derived macromolecules are involved in most biomineralization processes of particular interest in reactive bone induction with CaHap implants. From this practical significance, we have fundamentally examined the relationship between protein adsorption behavior and the structural feature of various kinds of synthetic hydroxyapatites (Haps), such as CaHap, strontium Hap (SrHap), calciumstrontium Hap (CaSrHap), and carbonate-containing CaHap (CAp) in our previous papers.8-13 We reported the significance of positively charged adsorption sites on the * To whom correspondence should be addressed. Fax: +81-72978-3394. E-mail:
[email protected] (1) Tiselius, A.; Hjerten, H.; Levin, O. Arch. Biochem. Biophys. 1956, 65, 132. (2) Hjerten, S. Biochim. Biophys. Acta 1959, 31, 216. (3) Kawasaki, T.; Niikura, M.; Takahashi, S.; Kobayashi, W. Biochem. Int. 1986, 13, 969. (4) Kawasaki, T.; Ikeda, K.; Takahashi, S.; Kuboki, Y. Eur. J. Biochem. 1986, 155, 249. (5) Kawasaki, T.; Kobayashi, W.; Ikeda, K.; Takahashi, S.; Honma, H. Eur. J. Biochem. 1986, 157, 291. (6) H. Wassell, D. T.; Hall R. C.; Embery, G. Biomaterials 1995, 16, 697. (7) Mayhall, C. W. Arch. Oral Biol. 1970, 15, 1327. (8) Kandori, K.; Sawai, S.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf. 1992, 68, 283. (9) Kandori, K.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf., A 1993, 80, 287. (10) Kandori, K.; Saito, M.; Saito, H.; Yasukawa, A.; Ishikawa, T. Colloids Surf., A 1995, 94, 225. (11) Kandori, K.; Saito, M.; Takebe, T.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1995, 174, 124.
exposed ac or bc crystal faces (called C-sites) for determining the saturated amounts of adsorbed bovine serum albumin (BSA) (nsBSA), acidic protein, in our previous communications.10-12 On the other hand, we found recently that lysozyme (LSZ), basic protein, has less adsorption capacity to CaHap, SrHap, and CaSrHap particles rather than BSA and the saturated amounts of adsorbed LSZ (nsLSZ) depend on neither the cation/PO4 molar ratio nor the number of C-sites of these particles.13 Therefore we proposed in this paper that the adsorption sites of LSZ molecules are phosphate ions between C-sites exposed on ac or bc faces. This difference in the adsorption capacity of BSA and LSZ to CaHap was further confirmed by desorption and preadsorption methods.14 More recently, we examined the adsorption of BSA and LSZ on oleyl phosphate grafted CaHap with various extents of surface hydrophobicities and found pronounced effects of the hydrophobic moiety of adsorbent on protein adsorption.15 On the contrary, since the most proteins contained in biofluids of interest, such as blood, are multicomponent systems, the interaction of the proteins may become important in the adsorption. In general, it has been found that preferential or selective adsorption occurs so that certain proteins may be enriched in the surface relative to the solution and vice versa. Nevertheless, information on time dependence of relative quantities adsorbed in multiprotein systems is largely missing. One of the few such studies is given by Gendreau et al.16 They found that for a 1:1 mixture of albumin and fibrinogen, albumin predominated in the first 7 min and then was gradually (12) Kandori, K.; Shimizu, T.; Yasukawa, A.; Ishikawa, T. Colloids Surf., B 1995, 5, 81. (13) Kandori, K.; Horigami, N.; Kobayashi, H.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1997, 191, 498. (14) Kandori, K.; Fujiwara, A.; Mukai, M.; Yasukawa, A.; Ishikawa, T. Colloids Surf., B 1998, 11, 313. (15) Kandori, K.; Mukai, M.; Fujiwara, A.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1999, 212, 600. (16) Gendreau, R. M.; Leininger, R. I.; Winters, S.; Jakobsen, R. J. In Biomaterials: Interfacial Phenomena and Applications; Cooper S. L., Peppas, N. A., Eds.; Advances in Chemistry Series No. 199; American Chemical Society: Washington, DC, 1982; p 371.
10.1021/la9906424 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/08/2000
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Figure 1. Transmission electron micrographs of CaHap-1 (A) and -2 (B) particles. Table 1. Properties of CaHap Particles Employed for Competitive Adsorption Experiments CaHap
Ca/PO4 molar ratio
1 2
1.59 1.57
particle size/nm length
width
crystallite size/nm
specific surface area/m2 g-1
71 46
18 20
99 66
88 89
displaced by fibrinogen on a variety of colloid surfaces. However, no systematic concurrent or competitive adsorption study of proteins onto CaHap is found in the literature except for the limited data on the dynamic adsorption behavior of human serum albumin onto CaHap.17 For these reasons, much of the effort should be focused on the adsorption behavior of mixed proteins. In the present paper, competitive and cooperative adsorptions of BSA and LSZ on CaHap particles were examined by kinetic and steady-state (isotherm) measurements. Understanding the mechanisms of competitive and cooperative adsorptions of proteins on CaHap is important for the development of biocompatible materials and adsorbents for separating proteins. Experimental Procedures Materials. Two kinds of colloidal CaHap particles with similar properties and shape, as displayed in Table 1 and Figure 1, were used. Clearly in Table 1, crystallite sizes estimated from the peak of the (002) face are close to the average length of each particle. Therefore, the CaHap-1 and -2 particles can be regarded as a single crystal. These particles were synthesized from a wet method by aging the precipitates, formed from the reaction of Ca(OH)2 solutions with H3PO4, in a Teflon vessel under CO2-free conditions at 100 °C for 48 h.13 The highly purified BSA (Mw 67 200 Da) and LSZ (Mw 14 600 Da) supplied from Sigma Co. were used as proteins. The isoelectric points of BSA and LSZ are 4.7 and 11.1, respectively. It should be noted that BSA is negatively charged but LSZ is positively charged at pH 6 employed in the protein adsorption experiments. Adsorption Experiment. The adsorption experiment was carried out in 10 cm3 Nalgen polypropylene (PP) centrifugation tubes in which varying concentrations of single protein or multiprotein solutions in deionized-distilled water of pH 6 were added to disperse the CaHap particles (100 mg) using the batch method given elsewhere.8 The initial concentrations of BSA and LSZ were varied 0-5 and 0-2 mg cm-3, respectively. The PP centrifugation tubes were gently rotated at 15 °C for 48 h in a thermostat. The concentrations of BSA and LSZ in the supernatant after centrifugation were determined separately by an (17) Aptel, J. D.; Thomann, J. M.; Voegel, J. C.; Bres, E. F. Colloids Surf. 1988, 32, 159.
Figure 2. Changes in electrophoretic mobility of CaHap-1 (O) and -2 (b) particles by pH. HPLC method using a YMC-Pack C4-AP column with UV monitoring at 220 nm.15 BSA and LSZ were separated efficiently on the column in the acetnitrile (AN)/water/trifluoroacetic acid (TFA) eluents. These proteins were eluted by increasing the AN content in the eluents with a linear gradient from 70/30/0.1 to 10/90/0.1 in AN/water/TFA composition and each protein showed one single peak. The protein concentrations were related to a calibration curve using the peak area as a measure. Preliminary experiments revealed a high reproducibility of the method using PP centrifugation tubes, and no protein adsorption to the PP tube could be detected. The electrophoretic mobility (em) of the protein-covered CaHap particles was measured by using an electrophoresis apparatus as reported before.8 In the kinetic study, the adsorption experiment was carried out as a function of time by changing the incubation time of the PP centrifugation tubes.
Results and Discussion Surface Charge of CaHap Particles. Prior to the adsorption experiments, we measured the surface charge of the CaHap particles because preferential adsorption of proteins on hydrophilic surfaces is generally governed by electrostatics.18 In Figure 2 are displayed the changes of em of CaHap-1 and -2 particles with pH of dispersion medium. The pH was adjusted by diluted KOH and HCl solutions. Both the particles show negative em values, (18) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267.
Adsorption of Proteins on Calcium Hydroxyapatites
Figure 3. Kinetics of adsorption of BSA and LSZ from single and mixed protein systems to CaHap-1: (O, b) BSA; (4, 2) LSZ. Open and filled symbols represent the data for single and mixed systems, respectively. Concentrations of BSA and LSZ are 5 and 1 mg cm-3, respectively.
and the negative surface charges of the particles are increased with increasing pH of the medium as well as usually observed for metal oxides and hydroxides. This fact proves that surface P-OH groups, as confirmed by FTIR spectra measured in vacuo though the data are not shown here, act as surface charge sites, and OH- and H+ ions are potential determining ions.11 It should be noted that both the CaHap particles are negatively charged at pH 6 where the adsorption experiments were carried out. Competitive Adsorption of BSA and LSZ on CaHap. The competitive adsorption of BSA and LSZ on CaHap-1 was examined by kinetic measurements. This measurement was performed from the mixed protein solution dissolving 5 mg cm-3 BSA and 1 mg cm-3 LSZ, corresponding to 1 in an LSZ/BSA molar ratio. Before considering the competitive adsorption behavior, we should take into account the agglomeration of proteins in the bulk solution. It is well-known that protein molecules form oligomers in the solution, so-called as quaternary structure, by their agglomeration even if they are homoproteins with the same sign and surface charge density. Therefore, it can be presumed that BSA and LSZ form complexes, BSA--LSZ+, through an electrostatic attraction in the solution because BSA and LSZ are oppositely charged, BSA- and LSZ+ at pH 6, as mentioned before. Unfortunately, the size, aggregation number, and charge ratio of the agglomerates of (BSA+-LSZ-) are obscure at the moment. Actually it is known that proteins form complexes which are agglomerates of two or more molecular units.19,20 The agglomerates may have a lower structural stability because BSA and LSZ molecules were completely separated by the high-performance liquid chromatography (HPLC) method as described above. The kinetic data of adsorption from single (open symbols) and mixed protein systems (closed symbols) are displayed in Figure 3. It is obvious that the adsorption rate of LSZ (4) is faster than that of BSA (O), though the saturated amount of adsorbed LSZ (nsLSZ) is less than that of BSA. At the initial stage, the protein adsorption kinetics is mainly controlled by the diffusion rates of the protein molecules rather than the electrostatic attraction and hydrophobic dehydration.18 Therefore, the fast adsorption rate of LSZ may be interpreted by the difference in the (19) Lansen, H. G. W.; Bargeman, D.; Bergveld, P.; Smolders, C. A.; Feijen, J. J. Colloid Interface Sci. 1984, 99, 1. (20) Brooks, D. E.; Greig, R. G. J. Colloid Interface Sci. 1981, 83, 661.
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molecular mass of these proteins; the diffusion rate (and hence their rate of arrival) to the CaHap surface of the smaller LSZ molecules is faster than that of the larger BSA. Since the (BSA--LSZ+) agglomerates may have less negative (or less positive) net charge than the large negative BSA single molecule, the adsorption capacity of the agglomerates to the negatively charged CaHap-1 particle is expected to be increased. The preferential adsorption of the larger associates has been, in fact, reported by Lansen et al.19 and Brooks and Greig.20 However, no change in the adsorption rates of BSA from single (O) and BSA-LSZ mixed systems (b) can be observed. This fact is due to the extremely high adsorption capacity of BSA to CaHap as confirmed in our previous studies.8-13 Since the adsorption capacity of LSZ to CaHap is low, the effect of LSZ in the agglomerates on the adsorption capacity of BSA to CaHap would be eliminated. BSA molecules strongly adsorb onto the CaHap-1 mainly through the electrostatic attractive force between the negative charge on BSA and the localized positive one on CaHap-1 originating from the C-sites, even though the net surface charge of CaHap-1 is negative at pH 6. This fact supports our previous finding on the important role of the C-site for adsorbing BSA onto CaHap.10-12 On the other hand, the adsorption rate of LSZ to CaHap is considerably reduced by coexistence of BSA (2). The nsLSZ value from the 1:1 mixture of BSA and LSZ is 20% less than that from the LSZ single system (4). This depression of adsorption rate of LSZ with addition of BSA can be explained by decrease the number of free LSZ molecules dissolving in a solution after formation of the (BSA--LSZ+) agglomerates. The free LSZ molecules would be captured by BSA ones to form the agglomerates. This result indicates that the adsorption of (BSA--LSZ+) agglomerates makes relatively small contributions to increase the nsLSZ at the LSZ/BSA molar ratio of 1. Another possibility of this depression can be considered as the preferential adsorption of BSA to positively charged C-sites on the CaHap-1 surface by an electrostatic attractive force. The preferentially adsorbed BSA molecules screen the adsorption sites of LSZ that are phosphate ions between C-sites.13 However, since the LSZ molecules should be adsorbed onto preadsorbed BSA ones by forming double protein adsorbed layers to raise the nsLSZ values,14 this mechanism would appear unlikely at the LSZ/BSA molar ratio of 1. Effects of BSA on the Adsorption of LSZ from BSA-LSZ Mixtures. Figure 4 shows the amounts of adsorbed LSZ on CaHap-2 from the BSA-LSZ mixtures in the presence of various amounts of BSA (A) and their em (B) as a function of equilibrium concentration of LSZ, equal to the total concentration of LSZ in solution. Figure 4C displays the amounts of adsorbed BSA for the systems with 2.5 and 5.0 mg cm-3 BSA. Unfortunately, the data for the systems with 7.5 and 10.0 mg cm-3 BSA could not be obtained by the HPLC method because of their high concentrations. In the absence of BSA, the adsorption isotherm of LSZ on CaHap-2 shows the pseudo-Langmuir type and em is reversed from a negative value to a positive one with adsorption of LSZ+ molecules as we reported before (b).8-12 On the other hand, the addition of BSA greatly affects the adsorption of LSZ as plotted by open symbols. In the presence of 2.5 mg cm-3 BSA (0), the amount of adsorbed LSZ is increased up to a LSZ concentration of 1.2 mg cm-3(marked by an arrow), corresponding to 2.2 in the LSZ/BSA molar ratio, and is decreased above the concentration exhibiting a maximum. In this system, the amounts of adsorbed BSA are simultaneously increased up to ca. 0.4 mg m-2 as depicted
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Figure 4. Adsorption isotherms of LSZ on CaHap-2 particles from BSA-LSZ mixtures in the presence of various amounts of BSA (A) and their em (B). The changes of the amounts of adsorbed BSA are displayed in (C). Concentration of BSA: (b) 0, (0) 2.5, (3) 5.0, (4) 7.5, and (O) 10.0 mg cm-3.
in Figure 4C, suggesting the cooperative adsorption behavior of LSZ with BSA. Indeed, the preferential adsorption of the large associates such as (BSA--LSZ+) agglomerates has been reported.19,20 The less negatively or less positively charged (BSA--LSZ+) agglomerates can more easily approach the negatively charged surface of CaHap-2 particles. This fact is further confirmed by the em measurement of the protein-covered CaHap-2 particles. The negative value of em closed to zero with increase in the adsorption of LSZ. Since the more positively charged (BSA--LSZ+) agglomerates can be produced at a high concentration of LSZ, it is presumed that the enhancement of LSZ adsorption will take place at a high LSZ region. However, the amount of adsorbed LSZ is decreased above the maximum point. Therefore, the decrease in the adsorption amounts of (BSA--LSZ+) agglomerates could be explained by increase in their size and/or by change in their surface hydrophobicity or conformational stability, that reduces the nsLSZ above the maximum. This subject
Kandori et al.
should be made clear in future investigations. The remarkable inhibition effect of BSA on LSZ adsorption is observed for the systems with 5 and 7.5 mg cm-3 BSA (3 and 4). The adsorption of LSZ was considerably depressed up to certain LSZ concentrations and increased linearly above these concentrations. The LSZ concentrations where the LSZ adsorption starts are 0.75 and 1.0 mg cm-3 LSZ in the systems with 5.0 and 7.5 mg cm-3 BSA, corresponding to the 0.7 and 0.6 in the LSZ/BSA molar ratio, respectively. The adsorption of BSA can be recognized in Figure 4C for the system with 5.0 mg cm-3 BSA and was saturated at ca. 0.75 mg cm-3 LSZ as indicated by an arrow, corresponding to the concentration where LSZ adsorption starts. This result suggests that LSZ molecules are captured in the agglomerates up to 0.6-0.7 in the LSZ/BSA molar ratio and are hard to be adsorbed on the CaHap-2 particle surface though BSA molecules are preferentially adsorbed, similar to the result observed at the competitive adsorption experiment in Figure 3. The adsorption of free LSZ molecules will take place after the adsorption of BSA was completed above these concentrations to form the double protein adsorption layer. The formation of a double protein adsorbed layer is confirmed by change in em in Figure 4B. The em values abruptly approach zero after the formation of double protein adsorbed layer as depicted by dotted lines in the figure. The strong inhibitory effect is further observed on the system with 10 mg cm-3 BSA (O). In this case, a number of added LSZ molecules are captured in the agglomerates and their adsorption is considerably suppressed over the whole LSZ concentration. Effects of LSZ on the Adsorption of BSA from BSA-LSZ Mixtures. Adsorption isotherms of BSA (A) onto CaHap-2 from the BSA-LSZ mixtures and em (B) of the particles in the presence of various amounts of LSZ, similar to Figure 4, are shown in Figure 5. The amounts of adsorbed LSZ in each system are shown in Figure 5C. In the absence of LSZ, the adsorption isotherm of BSA on CaHap-2 shows the pseudo-Langmuir type and negative em are increased with increase in the adsorption of negatively charged BSA molecules (b) as we reported before.13 The addition of LSZ exhibits a pronounced effect on the adsorption of BSA as shown in Figure 5A by open symbols. More BSA molecules are adsorbed from the BSALSZ mixtures rather than from the BSA single system, and the amount of adsorbed LSZ exhibits a maximum in each system containing 0.5, 1.0, and 1.5 mg cm-3 LSZ as marked by arrows. The increase of the amount of adsorbed BSA in the presence of LSZ is due to the formation of (BSA--LSZ+) agglomerates as same as the cooperative adsorption behavior observed for the adsorption of LSZ in the presence of 2.5 mg cm-3 BSA in Figure 4A. Indeed, the adsorption of 0.1-0.2 mg m-2 LSZ occurs in 0.5 and 1.0 mg cm-3 LSZ systems (Figure 5C) (0 and 3). The decrease of the BSA adsorption above the maximum would be due to the decrease of the LSZ/BSA molar ratio in a complex by increase in the BSA concentration. The agglomerates formed at low LSZ/BSA molar ratio (at high BSA concentration) will possess more large negative surface charge and prevent their adsorption to negatively charged CaHap-2 particles. The maximum cooperative adsorption behavior appears at the 1.5 mg cm-3 LSZ system (4) exhibiting a large amount of LSZ adsorption in Figure 5C. It is interesting that the maxima appear at 1.1, 2.2, and 3.3 mg cm-3 BSA in 0.5, 1, and 1.5 mg cm-3 LSZ systems, respectively, coinciding with the same LSZ/BSA molar ratio of 2.1. This fact implies that the maximum cooperative adsorption effect appears at the LSZ/BSA ratio of 2.1. The em values shift to positive by increasing the
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Figure 4A. The fact that the maxima appear at the same LSZ/BSA molar ratio supports the cooperative adsorption behavior of protein by formation of the (BSA--LSZ+) agglomerates. The size, electrical charge, hydrophobicity, and conformational stability of the (BSA--LSZ+) agglomerates formed at this molar ratio are optimum for the adsorption on the CaHap particles. Since the maximum would shift to a more high BSA concentration region, no maximum can be developed in the system with 2.0 mg cm-3 LSZ. Actually, the amount of adsorbed LSZ (O) is lower than that in the 1.5 mg cm-3 LSZ system over the whole BSA concentration (Figure 5C), while it increases rapidly with BSA concentration as though it attains a maximum above 5 mg cm-3 BSA. Conclusions The competitive and cooperative adsorption behaviors of BSA and LSZ on CaHap were compared by using a liquid chromatography method, and the following conclusions can be drawn. (1) The adsorption rate of LSZ was faster than that of BSA due to its larger diffusion coefficient, though the rate of BSA adsorption did not change by the addition of LSZ. (2) The adsorption of LSZ to CaHap is considerably reduced by coexistence of BSA that decreases the number of free LSZ molecules in a solution after the formation of (BSA--LSZ+) agglomerates. (3) A cooperative adsorption behavior of LSZ was observed on the BSA-LSZ mixtures in the presence of a lower concentration of BSA by the preferential adsorption of the larger (BSA--LSZ+) agglomerates. However, the adsorption of LSZ from the BSA-LSZ mixtures with higher concentrations of BSA was inhibited by capture of the LSZ molecules in the (BSA--LSZ+) agglomerates. After the preferential adsorption of BSA was completed, the adsorption of LSZ molecules took place to form the double protein adsorption layer. (4) The same cooperative adsorption behavior was observed on the adsorption of BSA in the presence of various amounts of LSZ. Figure 5. Adsorption isotherms of BSA on CaHap-2 particles from BSA-LSZ mixtures in the presence of various amounts of LSZ (A) and their em (B). The changes of the amounts of adsorbed LSZ are displayed in (C). Concentration of LSZ: (b) 0, (0) 0.5, (3) 1.0, (4) 1.5, and (O) 2.0 mg cm-3.
LSZ concentration, supporting the agglomerate formation mechanism. This molar ratio of 2.1 at the maximum is compatible with that of 2.2 observed for the system on the adsorption of LSZ in the presence of 2.5 mg cm-3 BSA in
Acknowledgment. The authors thank Mr. Masao Fukusumi of Osaka Municipal Technical Research Institute for help with the TEM observations. This work has been supported in part by a Grant-in-Aid for Scientific Research (C) of the Ministry of Education, Science, Sports and Culture, and Nippon Sheet Glass Foundation for Materials Science and Technology, and by The Cosmetology Research Foundation. LA9906424
