Calcite Crystal Growth Kinetics in the Presence of Charged Synthetic Polypeptides Branka Njegic´-Dzˇakula,† Ljerka Brecˇevic´,† Giuseppe Falini,‡ and Damir Kralj*,† Laboratory for Precipitation Processes, DiVision of Materials Chemistry, Ru{er BosˇkoVic´ Institute, P.O. Box 180, HR-10002 Zagreb, Croatia, and Dipartimento di Chimica “G. Ciamician”, UniVersita` di Bologna, Via Selmi 2, 40126 Bologna, Italy
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 5 2425–2434
ReceiVed December 10, 2008; ReVised Manuscript ReceiVed February 5, 2009
ABSTRACT: Poly-L-glutamic (pGlu) and poly-L-aspartic (pAsp) acids, as analogues of naturally occurring soluble acidic proteins involved in biomineralization processes, and poly-L-lysine (pLys), were used to investigate calcite growth kinetics as a function of the interaction between the charged polypeptides and the calcite surface. The kinetics of calcite crystal growth was determined in a simplified precipitation model system by inoculating well-defined calcite seed crystals into a moderately supersaturated solution containing one of the polypeptides. The parabolic rate law was found to be valid for the calcite crystal growth, the integration of ions into the spiral steps at the calcite crystal surface being the rate-determining mechanism. Small amounts of pGlu or pAsp caused an inhibition of calcite crystal growth, the effect being pAsp > pGlu, and the exponential dependence of the growth rate on supersaturation confirmed that surface nucleation was the growth controlling mechanism in the presence of the two acidic polypeptides. The pLys nonselective, weak, electrostatic adsorption at the crystal surface was probably responsible for increasing the calcite growth rate at low concentrations and for inhibiting it at higher concentrations. The strongest interactions between the crystal surfaces and the polypeptides were observed for the calcite/pAsp systems. They could account for coordinative interactions between the side chain carboxylic groups of the predominantly planar arrangement of the pAsp structure (β-pleated sheet) and Ca2+ ions from the calcite surface. Introduction Despite a considerable amount of research on biomineralization and bioinorganic materials,1-13 the mechanisms of the processes involved, especially of those occurring at the organic-inorganic interface, are still the subject of research. A number of studies14-16 suggest that in crystals growing from solution adsorption of organic (acidic) macromolecules on specific planes retards growth in the direction perpendicular to the plane. As the crystal grows, the adsorbed macromolecule gets occluded within the crystal structure. Its presence affects not only the crystal morphology but its mechanical properties as well. The mechanism of such interaction is suggested to be the result of stereochemical recognition and binding energy at the organic-inorganic interface.17 It has also been found that a selective precipitation of calcite and aragonite can be achieved in vitro by addition of acidic macromolecules, extracted from the calcitic or aragonitic layers of mollusc shells, and that the soluble proteins, rather than the insoluble part of the matrix, are responsible for the control of phase formation.17-21 However, the insoluble proteins in the matrix are believed to control the shape, size, and even aggregation of crystals.8,14,22,23 Many model studies on crystallization at the organic-inorganic interfaces have been carried out in vitro using the macromolecules extracted from biogenic crystals5,7,9,10,15,16,19,24-28 and the synthetic macromolecules4,5,8,29-31 considered to be analogues of biological macromolecules. However, of particular relevance are the atomic force microscopy (AFM) studies of the crystal growth kinetics of single calcite crystals in the presence of different Asp-oligopeptides and Asp-reach polypeptides, performed at a wide range of concentrations.32-34 As the authors suggested, the observed dual action of selected biomol* To whom correspondence should be addressed. Phone: (+385)-1-46 80 207. Fax: (+385)-1-46 80 098. E-mail:
[email protected]. † Ru{er Bosˇkovic´ Institute. ‡ Universita` di Bologna.
ecules, growth enhancement at low concentrations and inhibition at higher concentrations, could be attributed to their primary structure, that is, to the ionic charge and hydrophobicity. These findings contribute to the better understanding of the biomineralization process. In this work we investigated specific, coordinative interactions between the acidic polypeptides and mineral surfaces using poly-L-glutamic (pGlu) and poly-L-aspartic (pAsp) acids as analogues of naturally occurring soluble acidic proteins. From the kinetic data, we determined the mode and extent of calcite/polypeptide interactions. We assumed that the chemical-physical interactions between the organic substrate and the calcite crystals in biosystems, particularly the protein conformations, were similar to those between the calcite crystals suspended in electrolyte solutions and the synthetic polypeptide analogues adsorbed onto them. In order to find out whether conformity between the substrate and the adsorbed polypeptides, or the electrostatic interactions, have a decisive role in biomineralization poly-L-lysine (pLys) was also used in experiments. Poly-L-lysine is a basic polypeptide whose amino group side chains were positively charged in our experimental conditions. It was therefore to be expected that p-Lys would readily, although not specifically, attach itself to the negatively charged calcite surface. Bearing in mind the complexity of precipitation of sparingly soluble electrolytes, we used a simplified precipitation model system. The kinetics of crystal growth was determined by inoculation of a moderately supersaturated calcium carbonate solution containing one of the polypeptides with well-defined calcite seed crystals. In this way, it was possible to avoid nucleation and to discriminate clearly the growth process from nucleation. By comparative analysis of the main kinetic parameters (e.g., growth rate as a function of supersaturation), it was possible to specify the prevailing mechanism governing this particular process.35-39 The results are compared with similar investigations, particularly with the AFM studies of
10.1021/cg801338b CCC: $40.75 2009 American Chemical Society Published on Web 03/16/2009
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calcite single crystal growth in the presence of Asp-reach polypeptides. We hope that our results will contribute to the understanding of the role of stereochemical and structural matching between the synthetic analogues of acidic polypeptides involved in biomineralization, poly-L-glutamic acid and polyL-aspartic acid, and calcite crystals. Experimental Section Analytically pure chemicals, CaCl2 and Na2CO3 (both Merck), and high quality deionized water (conductivity RpGlu. A likely explanation for the observeddifferenceintheextent,aswellasinthephysical-chemical nature of the interactions between pAsp or pGlu and the calcite surface, could be found in their stereochemical difference. According to the predicted secondary pAsp and pGlu structures in aqueous solution, pAsp accounts for almost 50% of beta structures (β-strand and β-turn) and pGlu for about 50% of R-helix conformation.52 In a solution containing Ca2+ ions, and particularly when adsorbed on surfaces, pAsp tends to adopt more extended regions in β-pleated sheet conformation, while pGlu conserves R-helix and random coil conformations, and to a lesser extent the β-pleated sheet conformation of some distinct regions.22 The extended conformation that the polypeptide assumes in the β-sheet region favors the formation of more coordination bonds between its side chain carboxylate groups and calcium ions at the calcite surface. These chemical bonds can initiate the formation and stabilization of new crystalline {hkl} faces simply by preventing the formation and/or development of thermodynamically stable faces. The proposed model of strong coordinative interactions between pAsp and calcite surface is consistent and complementary to the described mechanism of functioning of the Asprich proteins, isolated from calcitic layer of mollusc shell, on that particular polymorph formation. At that, we keep in mind that, not only the stereochemistry but also the other parameters, such as the polypeptide’s sequence and structure accompanied with specific microenvironments, ionotropy, charge and the presence of specific insoluble macromolecules, can play a decisive role in production of a particular polymorph.27,28,53 The interactions between a positively charged basic polypeptide (such as pLys) and negatively charged calcite surface are most probably of electrostatic nature and incapable of stabilizing
Calcite Crystal Growth Kinetics
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Figure 8. Scanning electron micrographs of calcite crystals after overgrowth experiments in the presence of pAsp (A-C) and pGlu (D-F).
Figure 9. Scanning electron micrographs of calcite crystals after overgrowth experiments in the absence of additive (A-C) and in the presence of pLys (D-F).
specific new crystalline faces with respect to the energetically favored {104} ones. Conclusions (1) The crystal growth rates of calcite were determined for a range of initial supersaturations. At lower, S - 1 ) 6.1, as well as at higher, S - 1 ) 12.7, initial supersaturations, the parabolic rate law was valid, indicating that crystal growth was controlled by integration of ions into spiral steps at the crystal surface. (2) Addition of small amounts of pGlu or pAsp into the precipitation system (0.3 ppm < ci(pAsp, pGlu) < 2.05 ppm and 0.2 ppm < ci(pLys) < 7.0 ppm) caused the inhibition of calcite crystal growth, the pAsp effect being stronger than that of pGlu. The exponential dependence of the growth rate on supersaturation showed that surface nucleation was the rate controlling mechanism in the presence of both polypeptides. (3) Higher inhibitory effectiveness of pAsp might be explained by superior conformational matching between the calcium atoms at the calcite surface and the pAsp molecules in the partial β-pleated sheet, in contrast to the random coil conformation of pGlu.
(4) The addition of low pLys concentrations caused an increase in the calcite growth rate, whereas at higher pLys concentrations inhibition was noted. This was explained as a consequence of the pLys nonselective, weak, electrostatic adsorption at the crystal surface. (5) The strongest interactions between the crystal surfaces and polypeptides, observed for the calcite/pAsp systems, might be accounted for by coordinative interactions between the side chain carboxylic groups of the predominantly planar arrangement of the pAsp structure (β-pleated sheet), and Ca2+ ions from the calcite surface. (6) The observed structural matching between the calcite surface and the adsorbed pAsp, which served as a synthetic analogue of the naturally occurring hydrophilic macromolecules rich in aspartic and glutamic acid residues of certain biominerals, is believed to be of utmost importance for nucleation of calcite crystals in vitro and for their arrangement into the desired shape, size, and orientation. (7) The different behaviors of pAsp and pGlu may have additional relevance in understanding of the biomineralization processes. Aizenberg et al.54 reported that proteins rich in glutamic acid are able to stabilize amorphous calcium carbonate.
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Figure 10. Relative crystal growth rate of calcite seed in the presence of pAp, pGlu, and pLys, at 298 K and relative supersaturations, S - 1 ) 6.0 (9), S - 1 ) 5.5 (0), S - 1 ) 5.0 ([), and S - 1 ) 4.5 (]): ci(Ca2+) ) ci(CO32-) ) 1.0 × 10-3 mol dm-3.
In contrast, it has been demonstrated that aspartic rich proteins are usually involved in nucleation and crystal growth control. The results of the presented study confirm the higher propensity of pAsp to act as a crystal nucleator and modifier with respect to pGlu. Acknowledgment. The financial support from the Ministry of Science, Education and Sports of the Republic of Croatia (Project No. 098-0982904-2951) is gratefully acknowledged.
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