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Microcalorimetric Study of Protein Adsorption onto Calcium Hydroxyapatites Kazuhiko Kandori,* Kanae Murata, and Tatsuo Ishikawa School of Chemistry, Osaka UniVersity of Education, 4-698-1 Asahigaoka, Kashiwara, 582-8582 Osaka, Japan ReceiVed August 31, 2006. In Final Form: October 28, 2006 To clarify the adsorption mechanism of proteins onto calcium hydroxyapatite (Hap), the present study measured adsorption (∆Hads) and desorption (∆Hdes) enthalpies of bovine serum albumin (BSA; isoelectric point (iep) 4.7, molecular mass (Ms) 67200 Da, acidic protein), myoglobin (MGB; iep ) 7.0, Ms ) 17800 Da, neutral protein), and lysozyme (LSZ; iep ) 11.1, Ms ) 14600 Da, basic protein) onto Hap by a flow microcalorimeter (FMC). Five kinds of large platelike particles of CaHPO4‚2H2O (DCPD) after hydrolyzing at room temperature with different concentrations of NaOH aqueous solution ([NaOH]) for 1 h were used. DCPD converted completely to Hap after treatment at [NaOH] g 2%, and the crystallinity of Hap was increased with an increase in [NaOH] up to 10%. The amounts of protein adsorbed (∆nads) and desorbed (∆ndes) were measured simultaneously by monitoring the protein concentration downstream from the FMC with a UV detector. The ∆nads values were also measured statically by a batch method in each system. The ∆nads values measured by the FMC and static measurements fairly agreed with each other. Results revealed that BSA ∆HBSA ads was decreased with an increase in [NaOH]; in other words, ∆Hads was decreased with the improvement of Hap’s crystallinity, suggesting that the BSA adsorption readily proceeded onto Hap. This fact indicated a high affinity of Hap to protein. This affinity was further recognized by ∆HBSA des because its positive value was increased by increasing BSA [NaOH]. These opposite tendencies in ∆HBSA and ∆H revealed that Hap possessed a high adsorption affinity to ads des BSA (i.e., enthalpy facilitated protein adsorption but hindered its desorption). The fraction of BSA desorption was also decreased with an increase in [NaOH], confirming the high affinity of Hap to protein. Similar results were observed on the LSZ system, though the enthalpy values were smaller than those of BSA. In the case of neutral MGB, ∆HMGB ads also exhibited results similar to those of the BSA and LSZ systems. However, due to its weak adsorption by the van der Waals force, ∆HMGB des was small and almost zero at [NaOH] g 2%. Hence, the fraction of MGB desorption was less dependent on [NaOH].
Introduction Calcium hydroxyapatite [Ca10(PO4)6(OH)2, Hap] is in the space group P63/m; its unit cell parameters are a ) b ) 0.943 nm and c ) 0.688 nm, and it possesses two different binding sites (C and P sites) on the particle surface. Thus, it contains a multiplesite binding character for proteins.1-3 After dispersion of Hap particles in aqueous media, calcium atoms (C sites) are exposed on the Hap surface by dissolution of OH- ions at the particle surface. Therefore, the C sites arranged on the ac or bc particle face in a rectangular manner with the interdistance in the a or b directions equal to 0.943 nm and the interdistance in the c direction equal to 0.344 nm (c/2). The P sites are arranged hexagonally on the ab particle face with a minimal distance of 0.943 nm. The C sites are rich in calcium ions or positive charge and thus bind to acidic groups of proteins, but the P sites lack calcium ions or positive charge and therefore attach to basic groups of proteins. In practice, it is well-known that nonstoichiometric carbonated Hap is a major inorganic component of bone and tooth and possesses a high affinity for proteins.4 Hence, Hap is widely applied for separating various proteins in a highperformance liquid chromatography (HPLC) system. Many essential studies therefore have been reported.2,5,6 * To whom correspondence should be addressed: E-mail: kandori@ cc.osaka-kyoiku.ac.jp. (1) Kawasaki, T.; Takahashi, S.; Ikeda, K. Eur. J. Biochem. 1985, 152, 361371. (2) Kawasaki, T.; Niikura, M.; Takahashi, S.; Kobayashi, W. Biochem. Int. 1986, 13, 969-982. (3) Kawasaki, T.; Ikeda, K.; Takahashi, S.; Kuboki, Y. Eur. J. Biochem. 1986, 155, 249-257. (4) Kawasaki, T. J. Chromatogr. 1991, 544, 147-184.
In the past decade, we have conducted fundamental studies of the adsorptions of acidic bovine serum albumin (BSA), neutral myoglobin (MGB), and basic lysozyme (LSZ) onto various kinds of synthetic Hap particles.7-13 These studies clarified that the ) depends on the molar saturated amount of adsorbed BSA (nBSA s is ratio of Ca to phosphate (Ca/P) of the materials used. nBSA s increased with an increase in the Ca/P ratio, due to the electrostatic attractive forces between negatively charged BSA and the less negatively charged Haps with high Ca/P ratios at pH 6. On the contrary, the saturated amounts of adsorbed LSZ (nLSZ s ) on Hap particles is decreased with an increase in the Ca/P ratio, but no remarkable relationship with the Ca/P ratio was detected for the neutral protein MGB.12 Recently, our group also disclosed that the adsorption of BSA onto Hap is governed by Ca2+ ions complexing to BSA molecules (binding effect) together with the operation of C sites.14 However, this binding effect of Ca2+ ions is minor because the fraction of dissolution of surface Ca ions (5) Tiselius, A.; Hjerte´n, S.; Levin, O ¨ . Arch. Biochem. Phys. 1956, 65, 132155. (6) Thomann, J. M.; Mura, M. J.; Behr, S.; Aptel, J. D.; Schmitt, A.; Bres, E. F.; Voegel, J. C. Colloids Surf. 1989, 40, 293-305. (7) Kandori, K.; Sawai, S.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf. 1992, 68, 283-289. (8) Kandori, K.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf., A 1993, 80, 287-291. (9) Kandori, K.; Saito, M.; Saito, H.; Yasukawa, A.; Ishikawa, T. Colloids Surf., A 1995, 94, 225-230. (10) Kandori, K.; Saito, M.; Takebe, T.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1995, 174, 124-129. (11) Kandori, K.; Shimizu, T.; Yasukawa, A.; Ishikawa, T. Colloids Surf., B 1995, 5, 81-87. (12) Kandori, K.; Fudo, A.; Ishikawa, T. Phys. Chem. Chem. Phys. 2000, 2, 2015-2020. (13) Kandori, K.; Fudo, A.; Ishikawa, T. Colloids Surf., B 2002, 24, 145-153.
10.1021/la062562n CCC: $37.00 © 2007 American Chemical Society Published on Web 12/29/2006
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Table 1. Properties and Results of Protein Adsorption of Particles Produced by Hydrolyzing DCPD with Various Concentrations of NaOH Solutions 4nads (mg/m2) BSA
LSZ
MGB
[NaOH] (mass %)
specific surface area (m2/g)
Ca/P (atomic ratio)
FMC
static
FMC
static
FMC
static
0 1 2 6 10
5.6 27.2 96.6 118.9 80.8
0.97 1.32 1.52 1.56 1.57
2.4 2.2 0.6 0.3 0.7
2.8 1.4 0.6 0.3 1.1
0.4 0.5 0.1 0.2 0.3
a a 0.1 0.2 0.3
2.5 0.5 0.3 0.5 0.5
1.8 1.0 0.4 0.5 0.7
a
∆nads could not be measured.
of Hap is low (1.2-3.2%) in a neutral aqueous solution. Furthermore, since the number of functional groups is small, the binding effect of the counterions was only slightly detected on the adsorption of LSZ, even though the systems dissolve divalent (Ca2+, Ba2+) and trivalent (Al3+) ions. Despite these many investigations, the quantitative mechanisms and driving force of protein adsorption onto Hap particles still remain uncertain. Hence, we recently investigated protein adsorption behavior onto Hap particles using thermodynamic parameters (∆Gads, ∆Hads, and ∆Sads) determined by the van’t Hoff (VH) equation applied to the Langmuir isotherms measured at two different temperatures.15 We revealed that the adsorption processes of BSA and MGB with lower structural stability are driven by an entropy gain (i.e., these proteins adsorb onto the Hap surface accompanying a structural rearrangement). However, the adsorption of LSZ with relatively high structural stability is enthalpy-driven. Although the VH method is a convenient method for thermodynamic analysis, it provides questionable thermodynamic parameters because the irreversible nature of the protein adsorption process does not allow for such a thermodynamic analysis. Also the definition of standard states in the VH analysis is obscure, and adsorption is unlikely to be at the standard state. The obtained enthalpy (∆Hads) from the VH method may differ from that observed by direct microcalorimetric measurement. This phenomenon may occur also because of the heat capacity variations in temperature for most protein-ligand interaction systems. Therefore, to obtain enthalpy change during the protein adsorption process, microcalorimetric measurement is necessary. However, in the literature, calorimetric data regarding protein adsorption are scarce. Nyilas et al.16,17 and Filisko et al.18 examined the adsorption of blood proteins onto various surfaces, and the obtained results suggested that initially adsorbed protein undergoes more conformational change than a subsequent layer does. Resolving the integral adsorption heats of adsorption of albumin and ribonuclease onto polystyrene from a semiquantitative approach, Norde and Lyklema revealed that the heat associated with structural rearrangement upon adsorption is endothermic.19 They also found no predictable relationship between ∆Hads and charge on the particles or the substrate. Several investigators have obtained exothermic heats upon adsorption of protein onto a like-charged substrate, indicating that electrostatic attractive forces do not necessarily dominate spontaneous adsorption.20 Recently, Lee et al. reported precisely on the heat (14) Kandori, K.; Masunari, A.; Ishikawa, T. Calcif. Tissue Int. 2005, 76, 194-206. (15) Kandori, K.; Takaragi, S.; Murata, K.; Ishikawa, T. Adsorpt. Sci. Technol. 2005, 23, 791-800. (16) Nyilas, E.; Chiu, T-H.; Herzlinger, G. A. Trans.sAm. Soc. Artif. Intern. Organs 1974, 20, 480-490. (17) Nyilas, E.; Chiu, T-H.; Lederman, D. M. Trans.sAm. Soc. Artif. Intern. Organs 1976, 22, 498-513. (18) Filisko, F. E.; Malladi, D.; Barenberg, S. Biomaterials 1986, 7, 348-353. (19) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1979, 71, 350-366. (20) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 295-302.
of adsorption of LSZ onto polymeric substrates.21 They determined that endothermic discontinuities appear at rises between plateaus on the adsorption of LSZ onto polystyrene and poly(styrene-co-butylmethacrylate). They explained these endothermic discontinuities as a latent heat associated with a change to a more stable conformation after initial adsorption. To our knowledge, thermodynamics data on protein adsorption onto Hap are still lacking, particularly in terms of direct microcalorimetric measurement. The primary purpose of this study was to gain insight into the adsorption and desorption mechanism of proteins onto Hap through simultaneous measurements of enthalpy and amounts of protein adsorbed and desorbed by using a flow microcalorimeter (FMC) attached to a UV spectrometer. Since the few FMC studies of the protein adsorption system have been restricted to chromatographic supports with hydrophobic surfaces,22,23 this study on Hap will provide new information about protein adsorption onto the hydrophilic particles. The results will also offer further insight into the binding mechanism of protein adsorption onto Hap and contribute to the development of a high-quality HPLC column. Experimental Section Materials and Methods. Since fine particles may leak out through a filter in the FMC apparatus, the adsorbent particle must be larger than 1 µm. Thus, we prepared large Hap particles by hydrolyzing CaHPO4‚2H2O (DCPD; guaranteed reagent grade, Kanto Chemical) with different concentrations of NaOH aqueous solution (hereafter abbreviated as [NaOH]) at room temperature for 1 h, following the method reported by Ohta et al.24 The reaction can be written as 10CaHPO4‚2H2O f Ca10(PO4)6(OH)2 + 18H2O + 12H+ + 8PO43The particle size of DCPD was ca. 50 × 100 × 3 µm3. The hydrolysis reaction was performed by mixing 20 g of DCPD in a 0.5 dm3 NaOH aqueous solution. During the reaction, the solution was stirred with a magnetic stirrer. [NaOH] was varied from 0 to 10 mass % (Table 1). The precipitates obtained were filtrated and then washed with distilled water. After drying at 70 °C in air, the shape, specific surface area, crystal phase, and composition of the powders were determined by scanning electron microscopy (SEM), N2 adsorption measurements, X-ray diffraction (XRD), simultaneous thermogravimetry-differential thermal analysis (TG-DTA), and inductively coupled plasma atomic emission spectroscopy (ICPAES). The adsorption isotherm of N2 was measured at the boiling point of liquid nitrogen with the use of a computerized automatic volumetric apparatus built in-house. Prior to the measurement, the (21) Lee, V. A.; Craig, R. G.; Filisko, F. E.; Zand, R. J. Colloid Interface Sci. 2005, 288, 6-13. (22) King, M. A.; Cabral, A. C.; Queiroz, J. A.; Pinto, N. G. J. Chromatogr. 1999, 865, 111-122. (23) Thrash, Jr. M. E.; Pinto, N. G. J. Chromatogr. 2001, 908, 293-299. (24) Ohta, K.; Kikuchi, M.; Tanaka, J.; Eda, H. Key Eng. Mater. 2003, 240, 517-520.
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Kandori et al. consisting of two pairs of thermisters. Following the wetting, the syringe pump was turned on, and the adsorbent was equilibrated for 24 h with the carrier solution at a constant flow rate of 1 cm3/h from pump 1. After the cell temperature was equilibrated, the flow was switched to pump 2 with the same flow rate by means of the valve. Pump 2 supplied 1 × 10-4 mol/dm3 KCl with protein concentrations of 2 mg/cm3 BSA, 0.4 mg/ cm3 LSZ, and 1 mg/cm3 MGB. Since the rate of protein adsorption was slow, this adsorption experiment was measured for 17 h. After observation of the heat of protein adsorption (∆Hads), the same procedure was repeated by switching the valve to pump 1 to measure the heat of protein desorption (∆Hdes). A smooth and constant flow was achieved by using twin syringe pumps for the carrier and protein solutions. The quantity of adsorbed (or desorbed) protein molecules was also monitored by continuously measuring the concentration downstream from the particle bed with a UV detector at a wavelength of 280 nm. The detector signals for both the adsorption and desorption processes were stored in the computer, and the amounts of ∆nads and ∆ndes were determined by a frontal analysis method using computer software attached to the FMC apparatus. Representative experimental charts of FMC and UV are presented in Figure 2. The enthalpy change was calibrated with a known amount of electric current applied to the calibration heater (Figure 2). ∆nads and ∆ndes monitored by the UV detector were combined with the enthalpy change to calculate the heat of interaction per unit mole of proteins adsorbed or desorbed.
Results and Discussion Figure 1. Schematic diagram of the FMC apparatus. samples were evacuated at 100 °C for 2 h. All proteins were purchased from Sigma Co. (BSA, A-7030; MGB, M-0630; LSZ, L-6876). All other chemicals were purchased from Wako Pure Chemical, Ltd., and used without further purification. Protein Adsorption Isotherms Obtained under Batch Conditions. Since the FMC apparatus flows a protein solution through the particle bed, protein adsorption in the FMC system is a dynamic process. To compare the amounts of adsorbed proteins onto the particles, after treatment of DCPD powders with 0%, 1%, 2%, 6%, and 10% NaOH solution as listed in Table 1, in dynamic and static conditions, the amounts of proteins adsorbed in the static condition were measured by a batch method. This measurement was conducted at 25 °C employing a 1 × 10-4 mol/dm3 KCl solution of the protein in 10 cm3 Nalgen polypropylene (PP) centrifugation tubes, similar to the measurement we employed in previous studies.12-14 The PP tubes were gently rotated end-over-end at 25 °C for 48 h in a thermostat. The amounts of adsorbed protein were measured by the microburet method using a UV absorption band at 310 nm after centrifugation of the dispersions. Most of the UV experiments were triplicated and reproducible within 2%, indicating an uncertainty of 2 × 10-2 mg/m2 for the amounts of protein adsorbed. The ζ potential (zp) was also measured at 25 °C by an electrophoresis apparatus after redispersion of a small amount of the centrifuged Hap particles in the corresponding supernatant solution at 25 °C. Calorimetric Measurement. The enthalpy changes accompanying the adsorption and desorption of proteins onto Hap were measured by an FMC. A Mark-4Vi FMC (Microscal, Ltd.) was used at 25 °C. Figure 1 depicts a representative arrangement of this apparatus in a schematic diagram, reported elsewhere.25 All the materials contacting the measuring system were made of poly(tetrafluoroethylene). The sample cell of 0.17 cm3 was filled with 50 mg of particles. The sample was dried by evacuation in the cell at room temperature for several hours before the measurement. The purpose of this step was to remove air from the particle surface. Then a KCl solution of 1 × 10-4 mol/dm3 as the carrier liquid (pH 6.0) was introduced into the cell to prevent the dissolution of the particle surface and to keep the concentrations of K+ and Cl- ions constant during protein adsorption. The particles were wet with the evolution of heat of immersion detected with a bridge-type thermal sensor (25) Groszek, A. J.; Templer, M. J. CHEMTECH 1999, 11, 19-26.
Characterization of Hydrolyzed DCPD Particles. Figure 3 displays the SEM micrographs of DCPD after treatment at varied [NaOH]. The original DCPD particle has a smooth surface, while hydrolyzed DCPD particles have surface roughness made up of small rodlike particles. Ohta et al.24 reported that the hydrolysis of DCPD produces c-axis-oriented Hap aggregates on the particle surface: Hap crystallites grow in the (001) direction perpendicular to the surface of platelike DCPD particles. However, no such oriented crystal growth could be recognized in the present study. Figure 4 displays the XRD patterns of the particles depicted in Figure 3. The particle treated at 1% NaOH appears in weak patterns of Hap, together with those of DCPD. Characteristic patterns of Hap were observed after treatment of the particles at [NaOH] g 2%, indicating that DCPD converted completely to Hap. The Hap peaks sharpened with increasing [NaOH], suggesting improvement in the crystallinity of these Hap particles. This ready transformation of DCPD to Hap indicated that the large DCPD particle was porous despite its large size. However, the relative diffraction intensities of the 002 (2θ ) 25.9°) and 004 (2θ ) 53.2°; JCPDS 9-432) faces were not specifically large. This result did not agree with the results reported by Ohta et al.,24 suggesting again no production of c-axis-oriented Hap. TG measurement further supported the transformation of DCPD to Hap at [NaOH] g 2% (Figure 5). TG curves of DCPD had two sharp weight losses at 25-210 °C (21.2%) and 210-500 °C (6.3%). These weight losses were due to dehydration, as described by
CaHPO4‚2H2O f CaHPO4 + 2H2O (20.9%) 2CaHPO4 f Ca2P2O7 + H2O (6.6%). The values indicated in parentheses are the theoretical weight losses. The weight loss values measured in Figure 5 coincided with the theoretical ones. These two weight losses disappeared after treatment of the particles at [NaOH] g 2%, suggesting that the DCPD phase was completely transformed to Hap. In contrast, the Hap particles produced at [NaOH] g 2% had a small weight loss at 750-800 °C. Berry26 and Monma et al.27 reported that the weight loss from 25 to ca. 1000 °C observed for nonsto-
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Figure 2. FMC-UV charts of protein adsorption onto Hap particles produced from DCPD by hydrolysis with 10% NaOH solution for 2 h at 25 °C.
Figure 3. SEM micrographs of DCPD after treatment at various concentrations of NaOH.
ichiometric (Ca-deficient) Hap is caused by a release of adsorbed and bound H2O and small amounts of H2O formed by dehyroxylation of HPO42- ions:
2HPO42- f P2O74- + H2O (26) Berry, E. E. J. Inorg. Nucl. Chem. 1967, 29, 317-327.
The smaller weight loss at 750-800 °C is due to the reaction of the P2O74- ions with lattice OH- ions:
P2O74- + 2OH- f 2PO43- + H2O To confirm this consideration, we measured the Ca/P molar ratios (27) Monma, H.; Ueno, S.; Kanazawa, T. J. Chem. Technol. Biotechnol. 1981, 31, 15-24.
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Figure 4. XRD patterns of produced particles after treatment of DCPD at various concentrations of NaOH.
Figure 5. TG curves of produced particles after treatment of DCPD at various concentrations of NaOH.
of the particles by ICP measurement (Table 1). Clearly, the Ca/P molar ratios of Hap particles produced at [NaOH] g 2% ranged from 1.52 to 1.57, less than the theoretical value of 1.67. This result confirmed that the particles were Ca-deficient. The Ca/P ratio of DCPD was near unity. Protein Adsorption Isotherms Obtained under Batch Conditions. Prior to the FMC measurement, we measured the adsorption isotherms of proteins under static conditions. The adsorption isotherms and their ζ potential on the systems for BSA, LSZ, and MGB are indicated in Figure 6. All the adsorption isotherms showed the Langmuirian type, as they did in our previous studies.7-13 It should be noted that the adsorption isotherms of LSZ on the particles produced by hydrolyzing DCPD at [NaOH] ) 0% and 1% could not be measured due to their low amounts of adsorbed LSZ. The negative value of zp was increased by adsorption of negatively charged BSA, though the negative
Kandori et al.
zp reversed to a positive value by adsorption of positively charged LSZ. The MGB system exhibited almost constant zp in each particle. All the results of zp could be explained by the adsorption of electrostatically charged proteins. The amounts of adsorbed protein were in the order BSA > MGB > LSZ, coinciding with our previous studies.10-12 This result strongly confirmed that the positively charged C sites on Hap have an important role in the adsorption of acidic protein (BSA). Calorimetric Analysis of the Adsorption Process. The typical FMC and UV signals for protein adsorption onto Hap particles produced at [NaOH] ) 10% are indicated in Figure 2. It is easy to recognize that all of the adsorption and desorption processes of proteins exhibit endothermic enthalpy. The former process requires 8-16 h for equilibration, suggesting that protein adsorption onto calcium phosphate particles takes a long time. Similarly, the desorption process also requires 8 h for equilibration. Similar results were observed with the other systems. The adsorption and desorption peaks of proteins appear simultaneously on the UV chart (lower column in Figure 2), and their peaks correspond to the peaks of FMC. Table 1 summarizes the amounts of adsorbed proteins (∆nads), along with the ∆nads values measured under the static condition. The ∆nads values in the static condition were evaluated at the same protein concentration employed for the FMC measurement (denoted by the dotted line) in each adsorption isotherm in Figure 6. It is noteworthy that the ∆nads values obtained from the FMC and static methods fairly agreed with each other. This result allows us to discuss the protein adsorption behavior onto Hap using the enthalpy data obtained by the FMC. Figure 7 plots the values of ∆Hads, ∆Hdes, ∆nads, and ∆ndes obtained on the BSA, LSZ, and MGB systems by the FMC as a function of [NaOH]. Protein adsorption involves many subprocesses such as (i) changes in the state of hydration of the substrate and protein surfaces, (ii) rearrangements within the protein structure, and (iii) movement of ions into and out of the adsorbed layer. Microcalorimetric heats are the sums of heats generated by each subprocess. Norde and Lyklema concluded that the main enthalpy associated with structural rearrangement upon adsorption is large and endothermic.19,20 The ∆Hads values for all protein systems presented in Figure 7 exhibit endothermic heats. It can be concluded, therefore, that the spontaneous protein adsorption onto calcium phosphate particles is clearly entropydriven, most likely due to structural rearrangement. Of course, the substantial positive change in ∆Hads can also suggest the presence of significant electrostatic repulsive interactions. Since Hap possesses negative surface charge (Figure 6), this electrostatic repulsive interaction may affect the adsorption of negatively charged BSA. Indeed, the ∆Hads values for the BSA system are the largest among three proteins. Hence, the effect of this interaction cannot be neglected on the BSA system. On the other hand, the adsorption of positively charged LSZ at pH 6 could be driven by an electrostatic attractive force, accompanying negative ∆Hads (exothermic).26 However, the obtained experimental ∆Hads value was positive (endothermic). This opposite result confirmed that the adsorption of LSZ onto the Hap particle is not driven by an electrostatic attractive force. The structural rearrangement and/or orientation of LSZ are other possible adsorption modes. To confirm the protein adsorption mechanism, other studies have to be done, like the study of the variation of ∆Hads with the protein surface concentration and its comparison with adsorption isotherms. We can further calculate ∆Hads in mJ/m2 units by using ∆nads LSZ 2 as 2.9-0.16 mJ/m2 (∆HBSA ads ), 1.5-0.10 mJ/m (∆Hads ), and MGB 6.3-0.23 mJ/m2 (∆Hads ) for the DCPD particles after treat-
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Figure 6. Adsorption isotherms of proteins and their ζ potential for the particles produced from DCPD by hydrolysis with various concentrations of NaOH solution ([NaOH], mass %): (0) 0, (]) 1, (O) 2, (4) 6, and (b) 10.
Figure 7. ∆Hads, ∆Hdes, ∆nads, ∆ndes, and the fraction of desorption for three kinds of proteins as a function of [NaOH]: (O, 4, 0) adsorption, (b, 2, 9) desorption.
ment with 0-10% NaOH solution. The ∆Hads values of 2.9 mJ/m2 for BSA, 1.5 mJ/m2 for LSZ, and 6.3 mJ/m2 for MGB obtained on the original DCPD particles correspond to the literature values of 7-8 mJ/m2 for the integral heat of adsorption of ribonuclease through a structural rearrangement as reported by Norde and Lyklema.19 However, the magnitude of the ∆Hads values for Hap particles, produced by hydrolyzing DCPD powders
at [NaOH] g 2%, is smaller than that obtained for the original DCPD particles, indicating that Hap possesses a high affinity to protein as will be discussed below. The ∆Hads values for the Hap particle, after treatment of DCPD powders with 10% NaOH solution, measured by the FMC are 15 kJ/mol for BSA, 2 kJ/mol for LSZ, and 10 kJ/mol for MGB. However, the ∆Hads values calculated by the VH equation as
2070 Langmuir, Vol. 23, No. 4, 2007
reported in our previous paper15 are 59 kJ/mol for BSA, -59 kJ/mol for LSZ, and -17 kJ/mol for MGB. This difference in ∆Hads values, especially for the LSZ and MGB systems of those exhibiting opposite sign, would be due to the distinctions between FMC (dynamic) and batch (static) conditions and/or between Hap particles employed for measuring enthalpies. This result strongly suggests again a necessity of further work in the future. ∆HBSA ads decreased with an increase in [NaOH]; in other words, ∆HBSA ads was decreased with improvement of Hap’s crystallinity, suggesting that BSA adsorption readily proceeded. This fact indicated that the affinity of Hap to protein was high. Even though Hap possessed a high affinity to proteins, ∆nBSA ads decreased with an increase in [NaOH]. Since the surface roughness of the particles was increased with increasing [NaOH], the reduction of the effective surface area for protein adsorption seems to be a reason as reported for the adsorption behavior of proteins onto sintered Hap particles.28 The high affinity of Hap to protein was further recognized by ∆HBSA des , the positive value of which increased by increasing [NaOH], because the advancing endothermic process hindered BSA desorption. These opposite tendencies in ∆HBSA ads and ∆HBSA des revealed that Hap possessed a high adsorption affinity to BSA: enthalpy facilitated protein adsorption but hindered desorption. The fraction of BSA desorption, calculated BSA as the ∆nBSA des /∆nads ratio, further supported this result. The fraction of BSA desorption decreased with an increase in [NaOH], suggesting that the BSA adsorption became irreversible. The reduction of the fraction of BSA desorption might have induced small ∆Hdes values. The fact that the protein desorption process did not involve an irreversible denaturation was another reason for the small ∆Hdes values. Similar results could be seen with the LSZ system, although the enthalpy values were smaller than those of BSA. In the case of MGB, ∆HMGB ads also exhibited results (28) Rouahi, M.; Champion, E.; Gallet, O.; Jada, A.; Anselme, K. Colloids Surf., B 2006, 47, 10-19.
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similar to those of the BSA and LSZ systems. However, ∆HMGB des was small and almost zero at [NaOH] g 2%, probably due to the adsorption of neutral MGB molecules on phosphate ions exposed between C sites on the Hap surface by the weak van der Waals force. In this system, therefore, the fraction of MGB desorption showed less dependence on [NaOH]. Of course, the adsorption process was free-energy-driven but not enthalpydriven. This topic deserves further study in the future.
Conclusions The high affinity of Hap to protein, especially for the acidic protein BSA, was ascertained by measuring enthalpy through the FMC apparatus. ∆HBSA ads was decreased with improved crystallinity of Hap, suggesting that adsorption of BSA onto Hap readily proceeded. In contrast, the ∆HBSA des value was increased by increasing [NaOH]. These opposite trends of ∆HBSA ads and ∆ revealed that Hap possesses a high adsorption affinity to HBSA des BSA: enthalpy facilitates protein adsorption but hinders its desorption. The fraction of BSA desorption was also decreased with an increase in [NaOH], confirming the high affinity of Hap to protein. Similar results were obtained with the LSZ system, although the enthalpy values were smaller than those of BSA. In the case of neutral MGB as well as the BSA and LSZ systems, ∆HMGB ads was decreased with increasing [NaOH]. However, due to its weak adsorption by the van der Waals force, ∆HMGB des was small, approaching almost zero at [NaOH] g 2%. The fraction of MGB desorption was less dependent on [NaOH]. Acknowledgment. We thank Mr. Masao Fukusumi, Osaka Municipal Technical Research Institute, for help with the SEM observation. This work has been supported in part by a Grantin-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture. LA062562N
