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J. Phys. Chem. B 2008, 112, 2542-2547
Effects of Pyrophosphate Ions on Protein Adsorption onto Calcium Hydroxyapatite Kazuhiko Kandori,* Shohei Oda, and Shintaro Tsuyama School of Chemistry, Osaka UniVersity of Education, 4-698-1 Asahigaoka, Kashiwara, 582-8582 Osaka, Japan ReceiVed: August 9, 2007; In Final Form: NoVember 16, 2007
The effects of pyrophosphate ions (PP: P2O74-) on the adsorption of proteins onto calcium hydroxyapatite (Hap) were examined using typical proteins of bovine serum albumin (BSA: isoelectric point (iep) ) 4.7, molecular mass (Ms) ) 67 200 Da, acidic protein), myoglobin (MGB: iep ) 7.0, Ms ) 17 800 Da, neutral protein), and lysozyme (LSZ: iep ) 11.1, Ms ) 14 600 Da, basic protein). The UV and CD measurements determined that both the secondary and the tertiary structures of protein molecules do not vary in the presence of PP. The adsorption of BSA was strongly depressed by the addition of PP in all the methods with changing the order of PP addition. Even if BSA was pre-adsorbed on the Hap surface, PP replaced BSA molecules by strong preferential adsorption onto Hap to reduce the amounts of adsorbed BSA. A similar effect was observed with the adsorption of MGB. On the other hand, the amount of adsorbed LSZ (nLSZ) was increased with an increase in the concentration of PP, and the nLSZ value showed a maximum point in each adsorption isotherm. This fact was explained by a compression of the electric double layer (EDL) around each LSZ molecule by PP. This compression of the EDL induced the reduction of lateral electrostatic repulsions between charged LSZ molecules on the Hap surface and enhanced the formation of closed-packed monolayers to raise the nLSZ value. However, since the number of PPs around a LSZ molecule is decreased by an increase in the LSZ concentration in each system, the thickness of the EDL may be increased. Hence, nLSZ was reduced again after the maximum point in each system. Tripolyphosphate (TPP: P3O105-) ions exhibited similar effects on the adsorption behaviors of all proteins, but a much more pronounced effect was observed on the LSZ system. TPP with a higher eletronegativity shielded the EDL more highly than PP to increase the nLSZ value. The results of the zeta potential for all the protein systems supported the modes of protein adsorption discussed.
Introduction The interaction of protein molecules with inorganic materials has received much attention in many fields, such as biomineralization, biomaterials, biochemistry, biosensors, and the industry.1-3 It is well-known that calcium hydroxyapatite [Ca10(PO4)6(OH)2, Hap] is the major inorganic component of mammalian bones and teeth and that it possesses high affinity to the proteins. Hap is in the space group P63/m; its unit cell parameters are a equals b and is 0.943 nm and c is 0.688 nm, and it possesses two different binding sites (C and P sites) on the particle surface. Thus, it contains a multiple-site binding character for proteins.4-6 as is depicted a projection of the crystal structure of Hap in Figure 1. After dispersing Hap particles in aqueous media, calcium atoms (C sites) are exposed on the Hap surface by the dissolution of OH- ions at the particle surface. Therefore, the C sites, rich in calcium ions or positive charges to bind to acidic groups of proteins, are arranged on the ac or bc particle faces in a rectangular manner with interdistances of 0.943 and 0.344 nm (c/2) for the a (or b) and c directions, respectively. Indeed, Chen et al. reported that the -COO- claw of the protein grasps the calcium atoms of the Hap surface with its two oxygen atoms in a triangular form.7 The solid-state NMR study also revealed that the -COO- terminus of amelogenin is orientated to the Hap surface.8 The P sites, lacking calcium ions or positive charges attached to the basic groups of proteins, are arranged hexagonally on the ab particle face with a minimal distance of 0.943 nm. In addition, Hap is the most stable calcium phosphate under physiological conditions. Hence, Hap is widely * Corresponding author. E-mail:
[email protected].
applied for separating various proteins in a high-performance liquid chromatograph (HPLC) system. Many essential studies, therefore, have been reported on Hap.5,9,10 In the past decade, Kandori et al. have conducted fundamental studies on the adsorptions of acidic bovine serum albumin (BSA), neutral myoglobin (MGB), and basic lysozyme (LSZ) onto various kinds of synthetic Hap particles.11-19 On the other hand, recently, we examined the protein adsorption characteristics of Hap particles possessing anchored polyphosphate (APP: P-{O-PO(OH)}n-OH) branches on their surfaces and found interesting effects of the APP-branches on the protein adsorption behavior.20 The saturated amount of adsorbed BSA values was increased 3-fold by the formation of the APPbranches (i.e., BSA adsorption onto APP-Hap was enhanced by hydrogen bonding between oxygen and OH groups of the APP-branches and functional groups of BSA molecules). In the case of LSZ, the saturated amount of the LSZ value was increased 10-fold by introducing the APP-branches. This remarkable adsorption enhancement was explained by the threedimensional binding mechanism; LSZ molecules were trapped inside of the APP-branches. However, due to both their small size and their small numbers of functional groups of MGB, the saturated amount of MGB values did not vary. To clarify the structure of the APP-branches on the Hap surface, we examined the effects of pyrophosphate ions (PP: P2O74-) on the BSA adsorption behavior. In this comparison experiment, the BSA adsorption was depressed by the addition of PP. The effect of PP on the BSA adsorption was completely opposite to that of the APP-branches developed on the Hap surface. From this
10.1021/jp076421l CCC: $40.75 © 2008 American Chemical Society Published on Web 02/01/2008
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Figure 1. Projection of the crystal structure of Hap from perpendicular to the c axis.
result, therefore, we made the conclusion that the APP-branches are anchored and standing on the Hap surface. Since the binding affinity of the phosphate groups to the Hap surface is more than 20 times greater than that of carboxyl groups as reported by Moreno et al.,21 it can be expected that PP has, as a diphosphate, a high affinity to Hap. Uchiyama et al. reported that PP is an effective agent for the removal of pellicle from tooth surfaces.22 PP strongly adsorbs on the Hap surface and breaks the ionic bond between pellicle and Hap. Hexametaphosphate was also reported to be an active ingredient of toothpaste, and it has a great inhibitory activity in preventing the adsorption of chromogen.23 However, to the best of our knowledge, the effect of PP on the adsorption of proteins onto Hap was only reported by Rykke et al. using BSA.24 They used PP as an eluting agent in ion-exchange chromatography, but no experiment was performed on other proteins such as neutral and basic ones. Therefore, the mechanism of effects of PP on protein adsorption has not yet been fully understood. Hence, the aim of this study was to elucidate fundamentally this mechanism. The results obtained in the present study will contribute to the development of superb toothpaste and a highquality HPLC column. Experimental Procedures Materials and Methods. Colloidal Hap particles were prepared by the following wet method:11-15 0.405 mol of Ca(OH)2 was dissolved in 20 dm3 of deionized- and distilled water free of CO2 in a sealed Teflon vessel. After being stirred for 24 h at room temperature, 0.226 mol of H3PO4 was added into the solution, and the suspension was stirred for a further 24 h at room temperature. This suspension was aged in an air oven at 100 °C for 48 h. The Hap particles generated were filtered off, thoroughly washed with distilled water, and finally dried at 70 °C in an air oven for 24 h. All chemicals were reagent grade supplied from Wako Chemical Co. and were used without further purification. The shape, specific surface area, crystal phase, and Ca2+ and PO43- contents of the Hap particles were determined by transmission electron microscopy (TEM), N2 adsorption measurements, X-ray diffraction (XRD), and inductively coupled plasma atomic emission spectroscopy (ICP-AES). 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 samples
TABLE 1: Properties of Proteins no. of functional groups (per molecule) proteins BSA LSZ MGB
isoelectric molecular point weight (Da) size (nm) 4.7 11.1 7.0
67 200 14 600 17 800
4 × 14 3 × 3.5 3.5 × 4.5
-NH2
-COOH
680 155 34
680 32 36
were evacuated at 300 °C for 2 h. The XRD patterns were taken with Ni-filtered Cu KR radiation (40 kV and 120 A). The zeta potential (zp) of the particles was also measured by an electrophoresis apparatus. Protein Adsorption Measurements. The amounts of proteins adsorbed on the Hap particles were measured by a batch method as following the method employed in our previous papers16-19 under different orders of PP addition as will be described next. This measurement was conducted at 15 °C employing a 1 × 10-4 mol dm-3 KCl solution of the protein in 10 cm3 Nalgene polypropylene centrifugation tubes. The centrifugation tubes were gently rotated end-over-end at 15 °C for 48 h in a thermostat. The concentrations of the proteins were measured by the microbiuret method using an UV absorption band at 310 nm after centrifuging the dispersions. Most of the UV experiments were triplicated and were reproducible within 2%, indicating an uncertainty of 2 × 10-2 mg m-2 for the amounts of protein adsorbed. All proteins were purchased from Sigma Co. (BSA: A-7030, MGB: M-0630, and LSZ: L-6876). The properties of proteins used in this study are listed in Table 1. To clarify the effect of PP on the protein adsorption behavior, three different methods (1-3) were attempted by changing the order of PP addition as follows: (1) PP added simultaneously with BSA (PP + BSA), (2) BSA added followed by PP addition (PP-BSA), and (3) PP added after BSA was added (BSA-PP). Here, sodium pyrophosphate was used as a source of PP. The secondary materials in methods 2 and 3 were added after each system was equilibrated by the first ones at 15 °C for 24 h. For the comparison, adsorption experiments were examined using tripolyphosphate ions (TPP: P3O105-) instead of PP on method 1. UV and CD Spectra. The UV and CD spectral measurements were made at room temperature with a Hitachi UV spectrometer (UV-1200) and a CD spectropolarimeter (JASCO J-600), respectively. The protein solution was prepared by dissolving BSA, LSZ, and MGB in a 1 × 10-4 mol dm-3 KCl solution.
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Figure 2. TEM image of the Hap particle.
Figure 4. CD spectra of BSA, LSZ, and MGB solutions in 1 × 10-4 mol dm-3 KCl solution with different PP concentrations. [BSA] is 0.65 mg cm-3, [MGB] is 0.17 mg cm-3, and [LSZ] is 0.15 mg cm-3.
Results and Discussion
Figure 3. UV spectra of BSA, LSZ, and MGB solutions in 1 × 10-4 mol dm-3 KCl solution with different PP concentrations. [BSA] is 0.65 mg cm-3, [MGB] is 0.17 mg cm-3, and [LSZ] is 0.15 mg cm-3.
The UV and CD spectra at 190-340 nm of the near-UV region were measured using a quartz cuvette of 1 cm path length (for UV) and 0.1 cm path length (for CD). The spectra were scanned among 190 and 340 nm with 0.1 nm resolution; four scans were accumulated with a scan rate of 50 nm min-1 and a time constant of 0.125 s.
Properties of Hap Particles. The TEM image of the Hap particles used in this study is shown in Figure 2. The particles are rod-like and 15 × 60 nm2 in size. The specific surface area of the particles was 99.6 m2 g-1. From the N2 adsorption experiment, it was revealed that the particles are nonporous. The Ca/P molar ratio of Hap particles thus prepared was 1.58, suggesting that the particle is Ca2+-deficient. Secondary and Tertiary Structures of Protein Molecules. Prior to examining the effects of PP on the adsorption behavior of proteins onto Hap particles, we measured UV and CD spectra of proteins in a 1 × 10-4 mol dm-3 KCl solution with various amounts of PP to explore the conformational change of proteins by the addition of PP. Figure 3 displays the UV spectra of proteins measured at different contents of PP. It is clear from Figure 3 that there is no significant difference among all the systems examined, especially the baselines of the UV spectra at 300-350 nm are not varied, indicating that the protein molecules do not form molecular aggregates. Indeed, no precipitate and turbidity appeared in all the protein solutions with the addition of PP. These UV results suggest that PP does not affect the tertiary structure of the proteins. The residue ellipticity (θ) of proteins at the 190-250 nm region measured by the CD spectropolarimeter is shown in Figure 4. As expected, especially for BSA and MGB, these are predominately R-helical; the CD spectra shows a strong positive ellipticity at 190 nm and a negative ellipticity at 208 and 222 nm. On the other hand,
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Figure 5. Adsorption isotherms of BSA and its zp for three different orders of PP additions. [PP]: (b) 0, (O) 0.5, (0) 1.0, and (4) 1.5 mmol dm-3.
Figure 6. Adsorption isotherms of MGB and its zp at three different orders of PP additions. [PP]: (b) 0, (O) 0.5, (0) 1.0, and (4) 1.5 mmol dm-3.
due to its less R-helical conformation, a minor ellipticity is observed for LSZ. It is interesting that the intensities of these absorption peaks derived from the R-helical conformation were not changed by the addition of PP, suggesting that not only the tertiary structure but also the secondary structure do not vary in the presence of PP. The results of UV and CD spectra indicate that the effect of PP on the protein conformation is very weak. Effects of PP on Adsorption Behavior of Proteins onto Hap Particles. BSA. Adsorption isotherms of BSA onto Hap under three different orders of PP addition are shown in Figure 5 along with the zp. The adsorption isotherm of BSA from a 1 × 10-4 mol dm-3 KCl solution without PP (solid circles) is the Langmuirian type. The adsorption coverage of BSA, defined as the ratio of the experimental amounts of adsorbed BSA (nBSA) to the theoretical value, is 0.15. The latter value was estimated as 2.52 mg m-2 by assuming side-on adsorption of globular BSA molecules, which are prolate ellipsoids of 14 nm × 4 nm.
Since the solution pH of the system was ca. 8, the BSA molecules were negatively charged. Therefore, the negative values of the zp of this system were increased with an increase in the amount of adsorbed BSA. On the other hand, the adsorption of BSA was strongly depressed by the addition of PP. In method 1, the BSA adsorption was completely inhibited in the presence of 0.5 mmol dm-3 PP (open circles in Figure 5). In the case of method 2, PP pre-adsorbed on Hap at 1.0 mmol dm-3 completely inhibited the BSA adsorption. Particularly interesting results were observed using method 3. In this case, the addition of 1.0 mmol dm-3 PP (open squares) hindered the BSA adsorption as well as methods 1 and 2. This result indicates that PP adsorbs preferentially onto the Hap surface by desorbing BSA molecules pre-adsorbed at a first step; BSA molecules are replaced by PP. The preferential adsorption of PP on the C sites may reduce the adsorption of BSA. Yin et al. reported that the inhibition of BSA adsorption was achieved by the addition of PO43- ions.25 However, the concentration of
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Figure 7. Adsorption isotherms of LSZ and its zp at three different orders of PP additions. [PP]: (b) 0, (O) 0.05, (0) 0.1, and (4) 0.2 mmol dm-3.
PO43- ions that they examined was 50-100 mmol dm-3. The concentrations of PP observed in this study were almost 100 times smaller than that of the PO43- ions, indicating the high efficiency of PP. The large negative values of zp in the systems with PP for all the methods may support this preferential adsorption of PP. MGB. The similar inhibition effect of PP can be seen on the system of MGB adsorption as is depicted in Figure 6. The amounts of adsorbed MGB (nMGB) decreased with an increase in the concentration of PP for all methods. The PP molecules adsorb on the C sites and cover the phosphate groups between C sites, which are regarded as the adsorption sites of MGB,26 exposed on the ac and bc faces of the Hap particles. The absolute value of the negative zp decreased and became constant by progressing the MGB adsorption. Since MGB is a neutral protein with a low surface charge, this decrease of zp can be due to a shift of the slipping plane by MGB adsorption to develop a thick adsorbed layer. A constant negative zp value (-25 mV) was observed in the systems with 1.5 mmol dm-3 PP (triangles in Figure 6) with those exhibiting no adsorption of MGB regardless of the methods. This constant negative zp value can be regarded as the zp of Hap particles with saturated amounts of PP adsorbed. LSZ. A completely different adsorption behavior was observed by the addition of PP on the LSZ system as is displayed in Figure 7. Two remarkable features can be seen in Figure 7: (i) The amount of adsorbed LSZ (nLSZ) increased with an increase in the concentration of PP and (ii) the nLSZ value follows a gradual increase with the equilibrium concentration of LSZ (Ce) reaching a maximum value of adsorption at Ce equals 0.2 to ∼0.5 mg cm-3. At still higher Ce values, the isotherms show a tendency to decrease in adsorption. This trend becomes much more significant with an increase in the PP concentration. Since the cross-sectional area of PP is 0.5 nm2 molecule-1, the concentration of PP fully covers the surface of the Hap particles in the solution and amounts to 4.4 mmol dm-3. However, the concentration of PP examined in the present study was 0.5 to ∼0.2 mmol dm-3, 1.1 to ∼4.4% of the estimated value. This low concentration of PP denies the electrostatic adsorption of positively charged LSZ onto preferentially adsorbed negatively charged PP. In other words, the electrostatic attractive force
Figure 8. Adsorption isotherms of BSA, MGB, and LSZ in the presence of the same concentration of PP (O) and TPP (b) under method 1. (1 and 2) [PP] equals [TPP] and is 0.5 mmol dm-3 and (3) [PP] equals [TPP] and is 0.2 mmol dm-3.
between LSZ and PP is not a dominant factor in this case. As discussed before, since PP does not affect the secondary or
Protein Adsorption onto Calcium Hydroxyapatite tertiary structures of LSZ, we can determine that PP is an indifferent electrolyte. Hence, PP in the solution compresses the electric double layer (EDL) around each LSZ molecule. This compression of the EDL induces the reduction of lateral electrostatic repulsions between charged LSZ molecules on the Hap surface and enhances the formation of a close-packed monolayer to raise the nLSZ value. Therefore, a higher nLSZ value is achieved for systems with higher PP concentrations. However, since the number of PPs around a LSZ molecule is decreased by an increase in the LSZ concentration in each system, the thickness of the EDL may be increased. Hence, nLSZ was reduced again after the maximum point in each system. The adsorption of LSZ resulted in a polarity change of zp from negative to positive in methods 1-3, although the difference among the PP concentrations was small. This similar zp, irrespective of the methods, strongly reveals that the enhancement of LSZ adsorption occurs by the compression of the EDL but not by electrostatic attractive forces. Effects of Tripolyphoshate. Since the negativity of TPP ions is larger than the negativity of PP ions, a more intensive effect on the protein adsorption can be expected. To compare the effects of PP and TPP, the adsorption isotherms of BSA, LSZ, and MGB were measured at the same concentration of PP and TPP under method 1 and are displayed in Figure 8. Almost the same effect can be recognized in the BSA and MGB systems: the adsorptions of both proteins are similarly inhibited. In the case of LSZ, a similar trend can be seen for the PP and TPP systems, but the increase of the nLSZ value on the TPP system is much more pronounced than that on the PP system. This fact can be interpreted by the difference in the electronegativities between PP and TPP (i.e., TPP, with a higher eletronegativity, shields the EDL rather than PP). This result supports our explanation described before by regarding PP as an indifferent electrolyte. Conclusion The effects of PP ions on the adsorption of proteins onto Hap were examined using typical proteins BSA, MGB, and LSZ. The results of UV and CD measurements revealed that both the secondary and the tertiary structures of protein molecules do not vary in the presence of PP. The adsorption of BSA was strongly depressed in the presence of PP. Even if BSA was preadsorbed on the Hap surface, PP tore off the BSA molecules and replaced them using its strong preferential adsorption to reduce the amounts of adsorbed BSA. A similar effect was observed in the adsorption of MGB. On the other hand, the amount of adsorbed LSZ showed a maximum point in each adsorption isotherm. This trend became much more significant with an increase in the PP concentration. This fact was explained
J. Phys. Chem. B, Vol. 112, No. 8, 2008 2547 by changing the compression of the EDL around each LSZ molecule with PP. A similar effect was observed in the systems with TPP. Acknowledgment. Masao Fukusumi, Osaka Municipal Technical Research Institute, is thanked for help with the TEM observations. Drs. Kunio Takeda and Toshiko Moriyama at the Okayama University of Science are thanked for measuring the CD spectra. References and Notes (1) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110-115. (2) Ahn, E. S.; Gleason, N. J.; Nakahira, A.; Ying, J. Y. Nano Lett. 2001, 1, 149-155. (3) Habelitz, S.; Kullar, A.; Marshall, S. J.; DenBesten, P. K.; Balooch, M.; Marshall, G. W.; Li, W. J. Dent. Res. 2004, 83, 698-703. (4) Kawasaki, T.; Takahashi, S.; Ikeda, K. Eur. J. Biochem. 1985, 152, 361-371. (5) Kawasaki, T.; Niikura, M.; Takahashi, S.; Kobayashi, W. Biochem. Int. 1986, 13, 969-982. (6) Kawasaki, T.; Ikeda, K.; Takahashi, S.; Kuboki, Y. Eur J. Biochem. 1986, 155, 249-257. (7) Chen, X.; Wang, Q.; Shen, J.; Pan, H.; Wu, T. J. Phys. Chem. C 2007, 111, 1284-1290. (8) Shaw, W. J.; Campbell, A. A.; Paine, M. L.; Snead, M. L. J. Biol. Chem. 2004, 279, 40263-40266. (9) Tiselius, A.; Hjerte´n, S.; Levin, O ¨ . Arch. Biochem. Phys. 1956, 65, 132-155. (10) 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. (11) Kandori, K.; Sawai, S.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf. 1992, 68, 283-289. (12) Kandori, K.; Yamamoto, Y.; Saito, H.; Ishikawa, T. Colloids Surf., A 1993, 80, 287-291. (13) Kandori, K.; Saito, M.; Saito, H.; Yasukawa, A.; Ishikawa, T. Colloids Surf., A 1995, 94, 225-230. (14) Kandori, K.; Saito, M.; Takebe, T.; Yasukawa, A.; Ishikawa, T. J. Colloid Interface Sci. 1995, 174, 124-129. (15) Kandori, K.; Shimizu, T.; Yasukawa, A.; Ishikawa, T. Colloids Surf., B 1995, 5, 81-87. (16) Kandori, K.; Fudo, A.; Ishikawa, T. Phys. Chem. Chem. Phys. 2000, 2, 2015-2020. (17) Kandori, K.; Fudo, A.; Ishikawa, T. Colloids Surf., B 2002, 24, 145-153. (18) Kandori, K.; Masunari, A.; Ishikawa, T. Calcif. Tissue Int. 2005, 76, 194-206. (19) Kandori, K.; Murata, K.; Ishikawa, T. Langmuir 2007, 23, 20642070. (20) Kandori, K.; Tsuyama, S.; Tanaka, H.; Ishikawa, T. Colloids Surf., B 2007, 58, 98-104. (21) Moreno, E. C.; Kresak, M.; Hay, D. I. Calcif. Tissue Int. 1984, 36, 48-59. (22) Uchiyama, A.; Inoue, S.; Tanizawa, Y.; Ochiai, Y. J. Dent. Health 2004, 54, 132-140. (23) Gerlach, R. W.; Liu, H.; Prater, M. E. J. Clin. Dent. 2002, 13, 6-9. (24) Rykke, M.; Ro¨lla, G.; So¨nju, T. J. Dent. Res. 1988, 96, 517-522. (25) Yin, G.; Liu, Z.; Zhan, Z.; Ding, F.; Yuan, N. Chem. Eng. J. 2002, 87, 181-186. (26) Kandori, K.; Miyagawa, K.; Ishikawa, T. J. Colloid Interface Sci. 2004, 273, 406-413.
