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Inorganic Chemistry Communications 9 (2006) 164–166

Interaction of lysozyme during calcium carbonate precipitationat supramolecular level

Lin Yang, Lan She *, Jian-Guo Zhou, Ying Cao, Xiao-Ming Ma

College of Chemistry and Environmental Science, Henan Norma University, Xinxiang 453007, PR China

Received 1 November 2004; accepted 5 May 2005Available online 28 November 2005

Abstract

Interaction of lysozyme during calcium carbonate precipitation at supramolecular level was studied. The results showed that the ara-gonite phase of calcium carbonate was formed under the direction of lysozyme and the aragonite had also an important effect on thesecondary structure of lysozyme. These results may be explained by supramolecular interaction between lysozyme and CaCO3.� 2005 Elsevier B.V. All rights reserved.

Keywords: Supramolecular interaction; Biomineralization; Calcium carbonate; Lysozyme

Biological matrices in biological organisms are capableof controlling inorganic crystal growth to a remarkable de-gree during biomineralization [1,2]. Their complementarityto surface structure, charge and geometric configuration isone of the important aspect of this influence. Some mech-anisms of the effect of the organic matrices on the forma-tion of the crystals are understood [2–5]. But reports onthe influence of the formation of the crystals on the config-uration of the biological matrices are lacking, althoughMann [6] has predicted the existence of a mutual influencebetween the supramolecular organic matrices and the crys-tallization of the inorganic materials. The aim of our exper-iment was designed to study the mutual influence betweenlysozyme and the crystallization of calcium carbonate,especially the influence of the crystallization of calcium car-bonate on the configuration of lysozyme.

The crystallization of CaCO3 in lysozyme aqueous solu-tion was carried out as previously reported [7]. Pulverizedsodium carbonate (0.05 mol) and the same amount ofanhydrous calcium chloride were placed at the bottom ofa big beaker (250 ml) and a small beaker (100 ml), respec-tively, and the small beaker was placed into the big beaker.Then, lysozyme aqueous solution was slowly added into

1387-7003/$ - see front matter � 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.inoche.2005.05.026

* Corresponding author. Tel.: +863733328117; fax: +863733328507.E-mail address: shelan4100@hotmail.com (L. She).

the beakers until solution surface exceeded the inner smallbeaker wall by 5–6 mm and 0.05 M Tris–HCl was used asbuffer solution (pH 8.6). CaCO3 was formed because ofthe diffusion of Ca2+ and CO2�

3 . Control experiments wereperformed in the absence of lysozyme only using CaCl2 andNaCO3 solution as master solution. In the lysozyme bear-ing experiments, the mass of CaCl2 and NaCO3 were keptconstant (0.05 mol), while varying concentration of lyso-zyme (0.2%, 0.4% and 1.0%). Three parallel experimentswere performed simultaneously for every concentration oflysozyme solution. Free Ca2+ and CO2�

3 concentration insolution were determined by a PH/ISE meter (modelPXSJ-216) and titration method, respectively. The initialsupersaturation of the control and lysozyme bearing solu-tion were 2215 and 1857, respectively, which were calcu-lated using the EQPITZ program [8]. This observationsuggests that lysozyme favored the nucleation of the cal-cium carbonate, thus enable nucleation at lower values ofsupersaturation.

The obtained crystals were characterized by scanningelectron micrographs (SEM) surface morphology analysisand powder X-ray diffraction (XRD) analysis. In the caseof the different mass concentration of lysozyme solutionthe results were similar, so we take the lysozyme solutionwhich mass concentration was 0.4% for example. It canbe seen from the Fig. 1(a) that the crystalline products in

Fig. 1. SEM of the obtained crystals. (a) Calcium carbonate crystals formed in distilled water; (b) calcium carbonate crystals formed in aqueous solutionof the lysozyme.

Fig. 2. FT-IR spectra of the pure lysozyme and the CaCO3–lysozymesolution. (a) Pure lysozyme; (b) CaCO3–lysozyme solution; (c) CaCl2–lysozyme solution.

L. Yang et al. / Inorganic Chemistry Communications 9 (2006) 164–166 165

the distilled water were irregular, unsystematic and looselumps, and the XRD analysis proved that it were calcite.Fig. 1(b) showed a SEM of the products in the lysozymeaqueous solution. From Fig. 1(b), we can see that the crys-tals formed in the lysozyme solution were efflorescent bun-dles of needles. Powder XRD analysis indicated that thecrystal phase of the obtained crystal CaCO3 samples weremixture of aragonite and calcite. The fraction of aragonitein product was about 85% as determined by Rao equation[11]. These results indicated that the crystals formed in thelysozyme aqueous solution were mostly aragonite and lyso-zyme could direct the crystallization of calcium carbonateto form the aragonite.

In order to study the mechanism of the crystallization ofCaCO3 in the lysozyme solution, the solution of the reac-tive system was recorded on the FT-IR spectrophotometer.Because of the formation of the calcium carbonate crystalsin the reactive system, the solution should be a CaCO3–lysozyme solution after the reaction and the concentrationof calcium ion should be so low that the changes in theFT-IR spectra of lysozyme should be caused by CaCO3

crystals. It had been proved by the determination of con-ductivity and the concentration of the Ca2+ before andafter the reaction. In addition, the pure lysozyme, themixed solution of calcium chloride and lysozyme (CaCl2–lysozyme solution) and the mixture of sodium carbonateand lysozyme (NaCO3–lysozyme solution) were also re-corded on the FT-IR spectrophotometer.

The main frequency of the pure lysozyme, the solutionof the reactive system and the CaCl2–lysozyme solutionwere shown in Table 1, in which the bands in the 1630–1641 cm�1 region are assigned to the amide I band [12].In comparison with the IR spectrum of the pure lysozyme,the amide I band of the IR spectra of the CaCO3–lysozyme

Table 1The main frequencies of the pure lysozyme solution and the CaCO3–lysozyme solution in FT-IR spectra (cm�1)

Assignment Amide I band (cm�1)

Pure lysozyme 1658CaCO3–lysozyme solution 1647CaCl2–lysozyme solution 1638

solution and CaCl2–lysozyme solution shifted to a lowwavenumber about 11 and 20 cm�1, respectively (Fig. 2),while the spectrum of the lysozyme in the NaCO3–lyso-zyme solution did not change. The amide I band is thecharacteristic band of the C@O stretching vibrations. Theexact location of the amide I band depends on the natureof the hydrogen band involving the C@O and N–H moie-ties [9,11,12]. The shift of the amide I band may be causedby the interaction between the aragonite dissolved into thelysozyme solution and the C@O of lysozyme. The hydro-gen band may be change and break because of thisinteractions.

The study has indicated that the practical structure ofthe peptide bond of the protein is a resonance hybrid. Inthis resonance hybrid, the nitrogen atom has a positivecharge and the oxygen atom has a negative charge. Fromthe FT-IR of the CaCl2–lysozyme solution, the shift ofthe amide I band was stronger than that of the CaCO3–lysozyme solution. This may be caused by the electrostaticinteraction between the calcium ions and the oxygen atomsand this interaction was stronger than that between thearagonite and the oxygen atoms in the CaCO3–lysozymesolution.

Table 2Secondary-structural analyses of pure lysozyme and CaCO3–lysozymesolution

a-Helices b-Sheets b-Turns Random

m(cm�1) 1661–1681 1615–1698 1661–1681 1637–1645Pure lysozyme (%) 41.9 35.8 11.4 10.9CaCO3–lysozymesolution (%)

22.3 54.1 14.5 9.1

166 L. Yang et al. / Inorganic Chemistry Communications 9 (2006) 164–166

The FT-IR technology was an effective method to inves-tigate the change of the protein secondary structure and ithas been widely used [13]. In order to obtain detailed infor-mation about the change of the lysozyme secondary struc-ture, the shape of the amide I band of the pure lysozymeand the CaCO3–lysozyme solution were analyzed by deriv-atization, deconvolution and curve fitting techniques [10].Based on the band areas of the curve-fitting, the total per-centage of each secondary structure of the pure lysozyme,the CaCO3–lysozyme solution and the CaCl2–lysozymesolution were shown in Table 2. From Table 2, the a-heli-ces decreased about 19.6%, b-sheets increased about 18.3%for the CaCO3–lysozyme solution in comparison with thepure lysozyme, indicating that some of the a-helices chan-ged into b-sheets. In this solution, CaCO3 might combinewith the C@O of lysozyme according to the results of theFT-IR spectrum, resulting in the weakness and break ofthe hydrogen bond. Hence, some of the a-helix wasstretched and changed into b-sheet. This indicated thatthe calcium carbonate crystals can effect the lysozyme sec-ondary structure.

In order to confirm the effect of crystallization of cal-cium carbonate on lysozyme, the obtained aragonite crys-tals were dissolved into the lysozyme aqueous solution.Then, the mixed solution was characterized by FT-IRand analyzed by derivatization, deconvolution and curvefitting techniques. The result showed that the a-helices de-creased about 21.1% and the b-sheets increased about20.8% for this mixed CaCO3–lysozyme solution, which isconsistent with the forenamed result. This further provedthat there is a certain interaction between lysozyme andthe CaCO3, the essence of which was an electrostaticattraction between CaCO3 and the C@O of lysozyme,namely supramolecular interaction.

It has been known that when crystallizing from pureaqueous solution, calcite crystals nucleate mainly byhomogeneous nucleation: this means that, in this case,the interfacial specific energy (crystal/solution) reachesthe order of 100 erg cm�2. When lysozyme enters the

solution the scenario changes to that of heterogeneousnucleation: high molecular weight objects exhibit to thesolution their ‘‘surfaces’’ which are surely larger thanthe size of critical nuclei of calcium carbonate polymor-phs (built up by a few ‘‘molecules’’ for medium-highsupersaturations). The activation energy for aragonitenucleation can be lowered, since the adhesion energy be-tween the substrates (lysozyme molecules) and the nucle-ating crystals reduces their total surface energy, soincreasing their nucleating probability. It is understoodwhy aragonite nucleation can compete with that of cal-cite, even if aragonite is not the stable polymorph atroom temperature and pressure.

In conclusion, lysozyme can direct the nucleation andcrystallization of the calcium carbonate and CaCO3 alsohave a certain effect on lysozyme. There is a supramolecu-lar interaction between lysozyme and CaCO3 and theseresults are very significant for understanding of the mecha-nism of biomineralization in the organic life. Further inves-tigations are in progress.

Acknowledgements

We are grateful to the financial support of the NationNature Science Foundation of China (20371016) and theNatural Science Foundation of Henan Province(0311020100, 0311020100).

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