패각형성에서 나노복합체와 유기물과의 상관관계에 대한 연구

김용완, 최청송

서강대학교 화학공학과

A Study of Correlation between Organic Matrix and Nanocomposite

Material in Oyster Shell Formation

Yong-Wan Kim, Cheong-Song Choi

Department of Chemical Engineering, SoGang University

1. Introduction

The oyster shell consists of mineralized composites of calcium carbonate, the most common biomineral, and macromolecules in an exquisitely controlled manner[1], exhibiting exceptional nanoscale regularity and strength that could overcome the intrinsic brittleness of the crystals. The higher-order architecture results from the presence of organic matrix that creates defects in the perfect lattice which, in turn, enhance the fracture toughness by both absorbing stress and deviating the propagation of cracks along the cleavage planes of calcium carbonate[2]. It has been proposed that the interaction of proteins with specific inorganic crystal planes is governed by intrinsic stereochemical recognition of the macromolecules[3]. Recently some results showed the conformation-dependent control of calcite crystal growth by a peptide of defined secondary structure[4], and suggested the construction a polypeptide that could bind to a specific surface of a growing calcite crystal and alter its morphology would be possible.

In this study, we specifically discuss shell formation of oyster, Crassostrea Gigas, which is a composite of crystallographically highly ordered calcium carbonate with intracrystalline proteins. The investigation of the structural relationship between organic matrix calcium carbonate is the main subject. The approach taken was to extract quantitative information on the shell protein structure in vivo using Fourier self-deconvolution analysis of amide 1 band spectra[5] obtained from FTIR spectroscopy. And in order to establish the correlation between morphological modification and polymorphic behavior, the effect of secondary structure variations on the shell formation was studied from calcium carbonate formation in the presence of proteins extracted from within biominerals. Also structure and texture in shell were investigated by wide-angle X-ray diffractometer. In particular, features of the coherence length to mineral tissues were interesting subject to be investigated. The results provide evidence on the role of protein structure in the calcification of shell and the methods for understanding the principles that govern shell formation.

2. Experimental Procedure

Shells of Crassostrea Gigas were freshly collected, soaked in 5% NaOH, lightly scrubbed, and dried at room temperature. After completely dried, the shells were finely ground using a mill. The mineral-specific powders were dialyzed against 10□ of 2% acetic acid. The soluble proteins were dialyzed against four changes of double-distilled water at 4℃ over 2 days and then lyophilized. Lyophilizer, EYELA model FD-81, was used at a pressure of 0.004 Torr and a condenser temperature of -75℃.  Isolation of chitin from oyster shell was accomplished as followed[6].

The spectra were recorded using a MIDAC GRAMS Fourier transform infrared spectrometer with a resolution of 4 cm-1. The system was purged with dry N2 to reduce interfering water vapor IR absorption and verified no water contribution by measuring KBr pellets without protein. For intracrystalline protein intercalated within either calcite or aragonite, the subtraction was performed. To obtain the undisturbed proteins vibration spectra, each synthetic calcite and aragonite reference IR spectrum was subtracted from each mineral-specific layer, respectively. The subtracted spectra were analyzed by second derivatization, resolution enhanced Fourier self- deconvolution in the amide 1 region and Gaussian curve-fitting[7] using the ORIGIN 5.0. Second derivative spectra were smoothed with the 9 point Savitzky-Golay filter method. The number of components and their peak positions were determined by second derivatization and used as starting parameters[8]. The secondary structure contents were calculated from the area of the each Gaussian bands and their fraction of the total area in the amide 1 spectral region.The band assignment of these bands is based on previous studies of proteins by vibrational spectroscopy[9].

Each layer and the synthetic crystals were characterized by recording a RIGAKU horizontal powder X-ray diffractometer using CuK□ radiation via a rotating anode at 30kV and 20mA. The data were collected in steps of 0.05o/10 sec and the range of scattering angles(2□) from 15o to 110o. All measurements were carried out in □/2□ mode. The coherence length(CL) represents the average distance between imperfections in specific crystallographic directions. It can be determined from the width of the diffraction peak by using the Scherrer equation[10]: CL=k□/(cos□)(□), where □ is the wavelength, □ is the 'Bragg angle, k, shape factor, varies from 0.89 to 1.39, and □ is the halfwidth. In our setup, □=1.5405Å and k=0.9. The following {hkl} families of reflections were monitored: (202), (111), (012), (116), (104), (110), (018) and (113). In order to obtain statistically reliable patterns of crystal perfection, three symmetry-related reflections were measured and averaged within each family.

3. Results and Discussion

Protein, chitin and CaCO3 in the mineral form of calcite and aragonite are the major chemical components of the Shells of Crassostrea Gigas. Their characteristics vary as a function of the shell growth. The crystallographically highly oriented oyster shell was subdivided into four layers according to its mineralogy. The outer, center, inner layer which are composed of calcite(hexagonal system), and myostracum of aragonite (rhombohedral system) differ primarily with respect to structure and morphology. The calcitic layer consists of layers of lamella of calcite typically within 10 nm thick. In contrast, the aragonitic layer, a bunch of palisade, is about 10□ thick and 0.5-1□ wide. Morphological variations may reflect relative differences in carbonate concentration and the matrix sheets in forming a shell. Demineralization of the dried shell by acetic acid, dialysis, and subsequent quantification indicate that about 0.5% by weight of the total weight of the dried shell are soluble proteins and 0.2% chitin. Electron diffraction have shown that protein sheet consists of a chitin-like core sandwiched between two layers of protein as protein/polysaccharide networks, enabling complex large-scale composite shell to be fabricated. Also it is possible that the chitin may also play important roles interacting with the polyanionic aragonite-determining proteins as a framework in early stage of shell forming.

It has been assumed that the intracrystalline proteins are involved in limiting and modulating growth at each layer, and thus need to be secreted and directed to specific sites in a continuous modeling process at the submicron level. This concept could be verified using coherence length. Also from the XRD measurements, the degree of long-range order was calculated by investigating the coherence length of each layer and then compared to synthetic crystals. There is a marked difference between the coherence length of them as shown in table 1. Calcium Carbonate exhibited seven main peaks and each peak related to each crystal face. The peaks in calcite are (012) peak at 23.0o, (104) peak at 29.4o, (110) peak at 35.9o, (113) peak at 39.4o, (202) peak at 43.1o, (018) peak at 47.5o, (116) peak at 48.5o, and (111) peak in aragonite at 26.2o. Specifically, the degree of long-range ordering is much less for the crystals of each layer and even less for the center layer. Dimensional changes in the tissues would have the greater effect on the tissue cracking, reducing the matrix cracking stress. That is, the degree of ordering strongly depended on the coherence length. Higher coherence length caused higher crystallinity of the shell. This feature could be correlated with the fact that the intracrystalline proteins secreted from oyster are selectively adsorbed on calcium carbonate crystals according to molecular recognition or biological activity, and this cause them to develop into biologically-formed nanocomposite. So the intracrystalline proteins are thought to be located at boundaries between coherent blocks within the biominerals. In contrast, textural isotropy could not be detected in the synthetic crystals grown in the presence of shell proteins. This was then attributed to the poorly controlled environment of synthetic crystal growth, relative to the biogenic one. And we noted that the shell proteins in vivo are in the higher structural ordered state, and □ structure was adopted much contents to each shell layer. Also as the shell undergoes a change of calcium carbonate polymorphs from aragonite to calcite, significant alterations of the protein conformation with the denaturing of □-helix and □-structure in the aragonitic layer induces. These results may be applicable to synthetic strategy in the processing of highly ordered nanostructural material design.

4. References

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