Langmuir 2000, 16, 6739-6744
6739
Inhibition of Hydroxyapatite Formation in Aqueous Solutions by Amino Acids with Hydrophobic Side Groups S. Koutsopoulos and E. Dalas* Department of Chemistry, University of Patras, GR-26500 Patras, Greece Received January 18, 2000. In Final Form: May 16, 2000 Four natural amino acids with hydrophobic nonpolar side group (alanine, phenylalanine, proline, and methionine) were examined and their activity on hydroxyapatite, HAP, crystallization was assessed. The method used was the constant composition technique, at low supersaturation levels. Crystallization took place exclusively on well-characterized HAP seed crystals introduced in supersaturated solutions only with respect to the calcium phosphate salt. The fit of the experimental data to the Langmuir adsorption isotherm indicates an inhibition mechanism based upon molecular adsorption of the amino acids on the HAP crystal surface at active growth sites. The inhibitory effect was related to the adsorption affinity constant of each amino acid to the crystal surface, which depends on the nature of the amino acids’ side group.
Introduction The crystallization and dissolution of sparingly soluble calcium phosphates, (mainly HAP) have attracted the attention of several authors mainly, because of their participation in biological calcification processes of teeth and bone formation.1-4 HAP is present in many cases of undesirable calcification such as articular cartilage,5,6 renal and bladder stone formation,7 and atheromatic plaque.8-10 Depending on the supersaturation level, ionic strength, and pH, several calcium phosphate phases may be formed (i.e., in order of increasing solubility hydroxyapatite, [Ca5(PO4)3OH], tricalcium phosphate, [Ca3(PO4)3], octacalcium phosphate, [Ca4H(PO4)3‚2.5H2O], and dicalcium phosphate dihydrate, [CaHPO4‚2H2O]). Hydroxyapatite is the most stable calcium phosphate salt under physiological conditions and over the past years it has been the model compound for biomineralization studies. The influence of amino acids on hydroxyapatite crystallization has been the subject of several studies in the past.11,12 These studies which aimed to the elucidation of the mechanism of HAP crystallization in biological conditions were basically concerned with spontaneous precipitation, employing high supersaturation levels. In such experiments both nucleation and crystal growth occur simultaneously, while the supersaturation levels employed allowed the formation precursor phases during the precipitation procedure. This resulted in a low reproduc* Corresponding author. (1) Neuman, W. F.; Neuman, M. W. Chemical Dynamics of Bone Mineral; The University Press: Chicago, 1958; p 67. (2) Neuman, W. F. Bone Material and Calcification Mechanisms. In Fundamental and Clinical Bone Physiology; Urist, M. R. Ed.; J.B. Lippincott: Philadelphia, PA, 1980; p 83. (3) Dalas, E.; Koutsoukos, P. G. J. Chem. Soc., Faraday Trans. 1989, 85, 2465. (4) Christoffersen, J.; Christoffersen, M. R. J. Cryst. Growth 1981, 53, 42. (5) Boskey, A. L.; Bullogh, P. G. Scann. Electron. Microsc. 1984, 28, 511. (6) Gordon, G. V.; Villanueva, T.; Shumacher, H. R.; Gohel, V. J. Rheumatol. 1984, 11, 861. (7) Nancollas, G. H. J. Cryst. Growth 1977, 42, 185. (8) Tomazic, B. B.; Brown, W. E.; Schoen, F. J. J. Biomed. Mater. Res. 1994, 28, 35. (9) Schmid, K.; McSharry, W. O.; Pameijer, C. H.; Binette, J. P. Atherosclerosis 1980, 37, 199. (10) Koutsopoulos, S.; Kontogeorgou, A.; Petroheilos, J.; Dalas. E. J. Mater. Sci.: Mater. Med. 1998, 9, 421.
Table 1. pK Values for the Ionizing Groups of the Amino Acids with Hydrophobic Side Groups at 37 °C hydrophobic amino acid
pK1(a-COOH)
pK2(a-NH3+)
alanine phenylalanine proline methionine
2.37 2.17 1.94 2.28
9.55 9.00 10.33 8.92
ibility and lack of a real insight into the biological calcification process.7 The inhibitory effect of alanine, proline, phenyalanine, and methionine, i.e., the amino acids with hydrophobic nonpolar side groups,13 on the crystal growth of HAP at low supersaturation levels will be investigated. For this study, the constant composition technique was employed.14 This technique is advantageous compared to other methods used for studying problems concerned with nucleation, crystal growth, and dissolution of salts, mainly because the concentrations of the solution species are kept constant during the course of the experiment. This is secured by the stoichiometric addition of the reactants consumed. Amino acids are compounds of major importance for living organisms moving freely in blood circulation after digestion of proteins. Also, it has been proven that they enter into the cell environment by simple diffusion,15 and thus their concentration is controlled by physiological mechanisms. It is apparent that studies of such molecules of biological relevance, in HAP formation, can be related directly to important processes of desirable or pathological calcification. In the present work, the interaction of amino acids with nonpolar hydrophobic side groups with HAP will be examined and results of the investigation conducted will be reported. Comments will be made on the amino acids’ effect on HAP crystal growth and their affinity for the (11) Tanaka, H.; Miyajima, K.; Nakagaki, M.; Sahimabayashi, S. Chem. Pharmac. Bull. 1989, 37 (11), 2897. (12) Kresak, M.; Moreno, E. C.; Zahradnik, R. T.; Hay, D. I. J. Colloid Interface Sci. 1977, 59 (2), 283. (13) Lehninger, A. L. Biochemistry, 2nd ed.; Worth Publishing: New York, 1975. (14) Koutsoukos, P. G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. Soc. 1980, 102, 1553. (15) Meister, A. Biochemistry of Amino Acids, 2nd ed.; Academic Press Inc.: New York, 1965.
10.1021/la000057z CCC: $19.00 © 2000 American Chemical Society Published on Web 07/06/2000
6740
Langmuir, Vol. 16, No. 16, 2000
Koutsopoulos and Dalas
Table 2. Crystallization of HAP on HAP Seed Crystals in the Absence of Any Additive at pH 7.4, 37 °C, 0.15 M NaCl, Total Calcium (Cat):Total Phosphate (Pt) ) 1.67' ∆G (J/mol)
expt no.
Cat (10-4 mol/L)
HAP
TCP
OCP
DCPC
R (10-8 mol min-1 m-2)
S-253 S-307 S-317 S-314 S-318
5.0 4.0 3.5 3.0 2.5
-4.43 × 103 -3.94 × 103 -3.63 × 103 -3.29 × 103 -2.88 × 103
-3.99 × 102 1.63 × 102 5.00 × 102 8.90 × 102 1.35 × 102
1.28 × 101 5.04 × 102 7.99 × 102 1.14 × 103 1.55 × 103
3.56 × 103 4.12 × 103 4.45 × 103 4.85 × 103 5.31 × 103
11.30 7.44 6.25 3.67 2.63
Table 3. Crystallization of HAP on HAP Seed Crystals in the Presence of Alanine at pH ) 7.4, 37 °C, 0.15 M NaCl, Total Calcium (Cat):Total Phosphate (Pt) ) 1.67 expt no.
Ala (10-4 mol/L)
Cat (10-4 mol/L)
HAP
S-253 S-329 S-150 S-148 S-364 S-363 S-326 S-258 S-297 S-296 S-298
0 1.68 2.81 5.61 11.22 16.84 22.45 5.61 5.61 5.61 5.61
5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 3.5 3.0 2.5
-4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.42 × 103 -4.42 × 103 -3.93 × 103 -3.63 × 103 -3.28 × 103 -2.87 × 103
∆G (J/mol) TCP OCP -3.99 × 102 -3.99 × 102 -3.97 × 102 -3.95 × 102 -3.90 × 102 -3.86 × 102 -3.81 × 102 1.67 × 102 5.05 × 102 8.95 × 102 1.36 × 103
1.28 × 101 1.40 × 101 1.48 × 101 1.68 × 101 2.07 × 101 2.45 × 101 2.84 × 101 5.08 × 102 8.03 × 102 1.15 × 103 1.55 × 103
DCPD
R (10-8 mol min-1 m-2)
3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.57 × 103 3.57 × 103 3.58 × 103 4.12 × 103 4.46 × 103 4.85 × 103 5.31 × 103
11.30 10.84 10.34 9.51 9.45 8.99 8.30 5.58 4.57 2.95 2.27
Table 4. Crystallization of HAP on HAP Seed Crystals in the Presence of Proline at pH ) 7.4, 37 °C, 0.15 M NaCl, Total Calcium (Cat):Total Phosphate (Pt) ) 1.67 expt no.
Pro (10-4 mol/L)
Cat (10-4 mol/L)
HAP
S-322 S-397 S-453 S-396 S-394 S-395 S-330 S-331 S-399 S-333 S-334
0 1.30 2.61 4.34 8.69 13.03 17.38 4.34 4.34 4.34 4.34
5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 3.5 3.0 2.5
-4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.42 × 103 -4.42 × 103 -3.93 × 103 -3.63 × 103 -3.28 × 103 -2.87 × 103
HAP substrate as well as the relationship between their functional side groups and their retarding behavior. Experimental Section Crystal growth experiments were conducted in a 250 mL double-walled water-jacketed glass cell, thermostated at 37 °C, connected to a recirculating water bath, by using the constant composition technique. Continuous monitoring of pH was performed using a combined glass/Ag/AgCl electrode (Metrohm, 6.0202.100), equilibrated at 37 °C, standardized before and after each experiment with NBS standard buffer solutions.16 The reaction kinetics was observed with an appropriately modified pH-stat system (Radiometer pH-meter 26, Radiometer titrator 11, Auto-burette ABU1C). A pH change as much as 0.005 pH units which followed HAP precipitation, triggered the addition of titrants from two mechanically coupled burets of the controlling system. The solution contents of two burets were as follows: the first one had CaCl2 and NaCl, while the other KH2PO4 and KOH, at appropriate concentrations so that no change in the supersaturation level or the desired stoichiometry in the working solution occurred.17 As a result, the HAP crystallization took place under conditions of constancy of the supersaturation and the concentration of the reactants. The solution pH was adjusted to 7.40 by the slow addition of standard potassium hydroxide solution (Merck, Titrisol), ionic strength was 0.15 M in NaCl and the temperature was adjusted at 37 ( 0.1 °C. (16) Bates, R. G. Determination of pH. Theory and Practice, 2nd ed.; John Wiley and Sons: New York, 1973. (17) Amjad, Z.; Koutsoukos, P. G.; Nancollas, G. H. J. Colloid Interface Sci. 1984, 101, 250.
∆G (J/mol) TCP OCP -3.99 × 102 -3.98 × 102 -3.97 × 102 -3.96 × 102 -3.92 × 102 -3.89 × 102 -3.85 × 102 1.66 × 102 5.04 × 102 8.94 × 102 1.36 × 103
1.28 × 101 1.37 × 101 1.47 × 101 1.59 × 101 1.89 × 101 2.19 × 101 2.49 × 101 5.07 × 102 8.02 × 102 1.14 × 103 1.55 × 103
DCPD
R (10-8 mol min-1 m-2)
3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.57 × 103 3.57 × 103 4.12 × 103 4.46 × 103 4.85 × 103 5.31 × 103
11.30 10.46 10.17 10.25 8.30 8.16 7.83 6.70 3.93 2.50 2.20
Experimental conditions were chosen in order to physicochemically resemble the biological ones (i.e., the ionic strength was 0.15 M and the pH was equal to 7.40 both similar to that of the human blood). Special care was taken in order to give sufficient time to the solute amino acid to interact with the HAP crystal surface into the working solution environment. This was achieved by letting phosphates, HAP seed crystals, and the amino acid tested at the desired pH and ionic strength in coexistence prior to the introduction of the calcium ions which initiated the crystallization process. Following the addition of calcium ions, we delay the start of the experiment for 2-4 s until the pH value reaches the primary adjustment value of 7.4. Blank experiments with and without the preequilibration procedure gave the same initial rates within the experimental error. This is considered an improvement compared to classical constant composition method, because adsorption of the solute does not happen simultaneously with the crystal growth of the seed crystals. Thus, the phenomena mentioned above can be evaluated and appreciated separately.18 As a result, the reproducibility of the measured crystal growth rates of HAP was better that 2%. A magnetic rodlike stirring bar continuously stirred working solutions. High-purity gas nitrogen, presaturated with water, was bubbled through the solutions before and during the course of the experiment in order to exclude diluted carbon dioxide. Constancy of the calcium and phosphate reactants during the experiments (to within (3%) was verified by periodically withdrawn aliquots from the working solution. The samples taken (18) Koutsopoulos, S.; Demakopoulos, J.; Argiriou, X.; Dalas, E.; Klouras, N.; Spanos, N. Langmuir 1995, 11, 1831.
Hydroxyapatite Formation in Aqueous Solutions
Figure 1. Effect of amino acid concentrations on the HAP crystal growth rate.
Figure 2. Dependence of the rate of HAP crystallization in the absence (9) and in the presence (2) of 3.35 × 10-4 M of methionine on the relative solution supersaturation (pH 7.4, 37 °C, 0.15 M NaCl). were filtered through membrane filters (Millipore 0.22 `ım) and analyzed as described elsewhere.19 The solids removed by filtration were examined by scanning electron microscopy (JEOL GSM 5200), X-ray powder diffraction (Philips PW 1830/1840), infrared spectroscopy (KBr pellet method, FT-IR Perkin-Elmer 16-PC), specific surface area (multiple point BET method PerkinElmer Sorptometer 212 D), and chemical analysis (which gave a molar ratio of calcium to phosphates of 1.67 ( 0.02). In all experiments, stock solutions of calcium dichloride, potassium dihydrogen phosphate, and sodium chloride were prepared from the respective crystalline solids (reagent grade, Merck) using triply distilled CO2-free water. Their standardization has been described in details elsewhere.20,21 The amino acids were purchased from Sigma Chemicals and BDH Biochemicals (extra pure more than 99%, L-chiral configuration). Stereochemistry of amino acids is of critical importance because it is related to their adsorption behavior. Thus, only the L-form, which is present in physiological systems, was used in the crystallization experiments (at the experimental conditions applied did not happen racemization13).
Results and Discussion It is important to note that in the concentration range where all kinetic experiments were done all amino acids (19) Dalas, E.; Kallitsis, J.; Koutsoukos, P. G. Colloids Surf. 1991, 53, 197.
Langmuir, Vol. 16, No. 16, 2000 6741
Figure 3. Dependence of the rate of HAP crystallization in the absence (9) and in the presence (2) of 4.34 × 10-4 M proline on the relative solution supersaturation (pH 7.4, 37 °C, 0.15 M NaCl).
Figure 4. Dependence of the rate of HAP crystallization in the absence (9) and in the presence (b) of 3.03 × 10-4 M of phenylalanine on the relative solution supersaturation (pH 7.4, 37 °C, 0.15 M NaCl).
tested were completely soluble.15 In experiments done at different concentrations of seed crystals (between 50 and 200 mg/L), no change in the same initial crystal growth rates normalized per unit surface area was observed. This is indicative of the fact that crystallization occurred without any spontaneous or secondary precipitation. Higher concentrations of seed crystals were not used in the experiments because at the ionic strength applied (0.15 M in NaCl) solid particles suffered from aggregation. Also, changes in the stirring rate between 100 and 800 rpm had no effect on the growth rate, suggesting that the rate-determining step is not diffusion from the bulk solution to the crystal surface.22 The solution speciation of each experiment was calculated from the pH value, the equilibria constants and the expressions for the mass and charge balance. In all cases, dissociation constants and ion pair formation between the calcium ions and the amino acid tested were taken into account.23,24 Amino acids’ stability constants used were modified at 37 °C using the van’t Hoff equation (biblio(20) Tomson, M. B.; Nancollas, G. H. Science 1978, 200, 1059. (21) Dalas, E.; Koutsoukos, P. G. J. Chem. Soc., Faraday Trans. 1989, 85, 3159. (22) Nielsen, A. E.; Toft, J. M. J. Cryst. Growth 1984, 67, 278.
6742
Langmuir, Vol. 16, No. 16, 2000
Koutsopoulos and Dalas
Table 5. Crystallization of HAP on HAP Seed Crystals in the Presence of Phenylalanine at pH ) 7.4, 37 °C, 0.15 M NaCl, Total Calcium (Cat):Total Phosphate (Pt) ) 1.67 expt no.
Phe (10-4 mol/L)
Cat (10-4 mol/L)
HAP
S-377 S-416 S-417 S-418 S-419 S-420 S-421 S-259 S-422 S-260 S-423
0 0.61 1.51 3.03 6.05 9.08 12.11 3.03 3.03 3.03 3.03
5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 3.5 3.0 2.5
-4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -3.93 × 103 -3.63 × 103 -3.29 × 103 -2.87 × 103
∆G (J/mol) TCP OCP -3.99 × 102 -3.99 × 102 -3.98 × 102 -3.97 × 102 -3.94 × 102 -3.92 × 102 -3.90 × 102 1.65 × 102 5.02 × 102 8.93 × 102 1.36 × 103
1.28 × 101 1.33 × 101 1.39 × 101 1.50 × 101 1.71 × 101 1.92 × 101 2.13 × 101 5.06 × 102 8.01 × 102 1.14 × 103 1.55 × 103
DCPD
R (10-8 mol min-1 m-2)
3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.57 × 103 3.57 × 103 4.12 × 103 4.46 × 103 4.85 × 103 5.31 × 103
11.30 9.84 10.20 7.53 6.27 4.48 3.65 3.10 2.30 2.08 1.25
Table 6. Crystallization of HAP on HAP Seed Crystals in the Presence of Methionine at pH ) 7.4, 37 °C, 0.15 M NaCl, Total Calcium (Cat):Total Phosphate (Pt) ) 1.67 expt no.
Met (10-4 mol/L)
Cat (10-4 mol/L)
HAP
S-432 S-438 S-437 S-387 S-388 S-440 S-390 S-441 S-392 S-393 S-404 S-435
0 0.34 1.01 2.01 3.35 6.70 10.05 13.40 3.35 3.35 3.35 3.35
5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 3.5 3.0 2.5
-4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.42 × 103 -3.93 × 103 -3.63 × 103 -3.28 × 103 -2.87 × 103
∆G (J/mol) TCP OCP -3.99 × 102 -3.99 × 102 -3.99 × 102 -3.98 × 102 -3.96 × 102 -3.94 × 102 -3.91 × 102 -3.88 × 102 1.65 × 102 5.03 × 102 8.93 × 102 1.36 × 103
Table 7. Affinity Constants and Kinetic Order Values for Hydrophobic Amino Acids Inhibitors of HAP Crystal Growth hydrophobic amino acid
Kaff, 102 L/mol
kinetic order, n
alanine phenylalanine proline methionine
2.86 24.39 5.74 6.21
1.9 ( 0.1 2.0 ( 0.3 2.1 ( 0.2 2.0 ( 0.3
graphically given values were at 25 °C) (see Table 1). This system of equations was solved for the solution species, making successive approximations for the ionic strength.25 For this reason, a computer program software was developed.26 Ionic activity coefficients of a z-valent ionic species, yz, were obtained from the modification of the Debye-Hu¨ckel equation proposed by Davies:27
log yz ) -Az2
xIS - 0.3IS 1 + xIS
(1)
where IS is the ionic strength of the supersaturated solution, A is a constant equal to 0.5236 (L/mol)1/2 at 37 °C. The driving force for the crystallization process may be expressed as the Gibbs free energy of transfer from a metastable supersaturated solution to equilibrium where the solution is saturated.
∆G ) -
RT IP ln o (2) ν k
(2)
sp
(23) Martell, A. E.; Smith, R. M. Critical Stability Constants; Vol. 1: Amino Acids; Plenum Press: New York, 1974. (24) Greenstein, J. P.; Winitz, M. Chemistry of the Amino Acids; John Wiley & Sons: New York, 1961. (25) Nancollas, G. H. Interactions in Electrolyte Solutions; Elsevier: Amsterdam, 1966. (26) Koutsopoulos, S. Ph.D. Thesis, University of Patras, 1997.
1.28 × 101 1.31 × 101 1.35 × 101 1.42 × 101 1.52 × 101 1.75 × 101 1.99 × 101 2.22 × 101 5.07 × 102 8.02 × 102 1.14 × 103 1.55 × 103
DCPD
R (10-8 mol min-1 m-2)
3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.57 × 103 3.57 × 103 4.12 × 103 4.46 × 103 4.85 × 103 5.31 × 103
11.30 11.07 10.20 10.15 8.54 7.68 7.40 6.30 3.64 2.46 2.58 1.51
in eq 2, IP is the ionic activity product and kosp the thermodynamic solubility product of the solid phase precipitated. R and T are the ideal gas constant and the absolute temperature respectively, while ν is the number of ions in the formula unit of the corresponding calcium phosphate, (e.g., ν ) 9 for HAP). Negative ∆G values represent solutions supersaturated with respect to the calcium phosphate solid under consideration and thus its formation is thermodynamically favorable. The experimental conditions for the precipitation of hydroxyapatite both in the presence and in the absence of hydrophobic amino acids inhibitors are summarized in Tables 2-5. The dependence of the crystal growth rate upon the amino acid concentration in the working solution is plotted in Figure 1. As may be seen from the Tables 2-5, changes in Gibbs free energy due to the presence of the amino acids are negligible. From this we may conclude that the decrease in the crystal growth rates are not due to reduction of the solution supersaturation as a consequence of ion pair formation between the amino acid and the reactants of the crystallization reaction. Neither inhibitor had any effect with respect to the calcium phosphate precipitating solid phase, which in all cases identified as hydroxyapatite, as revealed from stoichiometric chemical analysis and photographs from a scanning electron microscope. The crystal growth rates were obtained from numerical differentiation of the initial part of the curve, representing the total titrant volume added in the working solution with time. The data were corrected for changes in surface area during the crystallization process.28 All amino acids tested seemed to have an inhibitory effect on the hydroxyapatite crystallization process. The rates of crystallization of HAP were also found to depend on the solution (27) Davies, C. W. Ion Association; Butterworths: London, 1962. (28) Hohl, H.; Koutsoukos, P. G.; Nancollas, G. H. J. Cryst. Growth 1982, 57, 325.
Hydroxyapatite Formation in Aqueous Solutions
Langmuir, Vol. 16, No. 16, 2000 6743
Figure 5. Dependence of the rate of HAP crystallization in the absence (9) and in the presence (b) of 5.6 × 10-4 M alamine on the relative solution supersaturation (pH 7.4, 37 °C, 0.15 M NaCl).
supersaturation. The relative solution supersaturation of HAP, σHAP, is defined by
σHAP ) SHAP - 1 )
( ) IPHAP
kosp ,HAP
1/9
-1
(3)
According to the analysis of Nielsen, crystal growth rates depend on the relative solution supersaturation, σ, and thus the kinetic data were fitted to eq 4:29
R ) ksσn
(4)
where R is the overall crystal growth rate, k the rate constant, s a function of the number of the active growth sites on the crystal surface, and n the apparent order of the reaction. Logarithmic plots according to eq 4 are shown in Figures 2-5. From the linear plots obtained it was revealed that in all cases of HAP crystallization, both in the absence and in the presence of the additive, the apparent order, n, had a value of about 2, as shown in Table 5. This suggested a surface controlled spiral growth mechanism, which remained unchanged in the presence of the hydrophobic amino acids.30 Assuming a simple Langmuir isotherm for the adsorption of the hydrophobic amino acids on HAP crystal surface, a plot of Ro/(Ro - Ri), as a function of the inverse of the amino acid concentration in the working solution should, according to eq 5, yield a linear relationship. Ro and Ri are the rates of HAP crystallization in the absence and in the presence of the amino acids, respectively.
Ro 1 1 )1+ Ro - Ri kaff ceq
(5)
where kaff is the affinity constant of the adsorbate for the adsorbent. As may be seen in Figure 6, a straight line was obtained for all cases of hydrophobic amino acids, suggesting the validity of the assumed model. The affinity constants, as determined from the slope of the linear plots according to eq 5, are shown in Table 6. The excellent linearity of the plots suggest that the inhibitory action of the hydrophobic amino acids may interpreted in terms of (29) Nielsen, A. E. Pure Appl. Chem. 1981, 53, 2025. (30) Nancollas, G. H. In Biomineralization; Mann, S., Webb, J., Williams, R. J. P. Eds.; VCH Verlagsgesellschaft: Weinheim, F. R. Germany, 1989; Chapter 6, pp 156-187.
Figure 6. Kinetics of HAP crystal growth in the presence of various concentrations of amino acids with hydrophobic side groups according to the Langmuir-type kinetic model; 37 °C, pH 7.40.
their adsorption at the active growth sites on hydroxyapatite surface. The adsorption seems to follow a Langmuir type isotherm. The affinity constant is a measure of the affinity of the solute for the surface of the adsorbate. A high affinity constant value implies an extended adsorption. Generally speaking, solute species having functional groups such as carboxyl and/or amino groups are subject to reversible adsorption on a charged substrate. Amino acids with hydrophobic side groups were found to have a low adsorption affinity toward the HAP surface among amino acids.11 This was explained on the basis of the fact that amino and carboxylic groups bonded to the R carbon atom only slightly interact with the substrate, basically because of stereochemical configuration reasons. In addition, at the pH range where the kinetic experiments were performed (i.e., pH ) 7.40) the amino acids tested had a zero net charge. Thus, no electrostatic interaction was expected with the positively and negatively charged sites on HAP surface. As may be seen in Table 6, phenylalanine has the strongest affinity constant among hydrophobic amino acids. This fact is in accordance with the kinetic data. As may be seen in Figure 1, phenylalanine is an effective inhibitor of HAP crystal formation even at low concentration levels. Extensive adsorption and subsequent coverage of the crystal surface may explain its action. This is favored through the aromatic ring of phenyalanine’s side group, which is flat due to its aromatic character (π-bonding). It is also suggested that the ring lays down on the surface, possibly acting as an electron π-donor, which result in a weak bond between the ring and the HAP surface. This is in agreement with the molecule geometry while a similar explanation has been given for other inhibitors having aromatic rings.31-32 Alanine has the lowest affinity constant as may be seen from Table 6, which implies that it has narrow adsorption efficiency for the HAP crystal surface. From the literature there is evidence that alanine’s adsorption on HAP is nondetectable.11 This is in agreement with personal adsorption experiments, providing a plausible explanation (31) Giacovazzo, C.; Monaco, H. L.; Viterbo, D.; Scordari, F.; Gilli, G.; Zanotti, G. Catti, M. Fundamentals of Crystallography; Giacovazzo, C. Ed.; Oxford University Press: Oxford, UK, 1992. (32) Koutsoukos, P. G. Ph.D. Thesis, State University of New York at Buffalo, NY, 1980.
6744
Langmuir, Vol. 16, No. 16, 2000
of the low effectiveness upon the decrease of HAP crystal growth rate. It is important to note that the hydrophobic amino acids tested as possible inhibitors of HAP crystallization were active at concentration levels of the same order of magnitude with their physiological value at blood plasma,
Koutsopoulos and Dalas
(e.g., for alanine 4 × 10-4 mol/L, for phenylalanine 0.5 × 10-4 mol/L, for proline 2 × 10-4 mol/L, and for methionine 0.2 × 10-4 mol/L33). LA000057Z (33) Scriver, C. R.; Rosenberg, L. E. Amino Acid Metabolism and its Disorders; W. B. Saunders and Co.: Philadelphia, PA, 1993.
