Shell microstructure, mineralogy and in vitro crystallization studies on the shell soluble matrix of abalone, Haliotis discus hannai Ino.

ABSTRACT The shell microstructure of the abalone, Haliotis discus hannai Ino, was studied using scanning electron microscopy. The shell consists of the periostracum, prismatic, and nacreous layers with calcite in the outer prismatic layer and aragonite in the inner nacreous layer. FTIR analysis of the prismatic powder showed characteristic spectra of calcite: 876 [cm.sup.-1] (v2) and 710 [cm.sup.-1] (v4), whereas aragonitic nacreous powder had characteristic spectra of 858 [cm.sup.-1] (v2) and 712 - 700 [cm.sup.-1] (v4). Another absorption area in the range 2520-2650 [cm.sup.-1] in both nacreous and prismatic layers was assigned to HC[O.sub.3.sup.-] groups, residing in the mineral or at the organic mineral interface, which suggests a potential participation of HC[O.sub.3.sub.-] groups as an intermediate in shell formation. In the in vitro crystallization experiment, it was found that the soluble matrix from different shell layers were able to induce different crystals with special morphologies. Soluble matrix, isolated from the prismatic layer, was shown to induce globular calcite formation in vitro, whereas soluble matrix from the nacreous layer induced the formation of almost symmetrical, hedgehog-like, aragonitic aggregates. The regulating mechanism of the formation, morphologic development, and crystallography of minerals in abalone shell is discussed.

KEY WORDS: Haliotis discus hannai, shell, microstructure, mineralogy, soluble matrix, in vitro crystallization

INTRODUCTION

Mollusk shells are polycrystalline composites of calcium carbonate and ~1% to 5%, by weight, of proteins and glycoproteins (Kaplan 1998). The shell has a fracture strength of about 3000 times higher than that of the inorganic CaC[O.sub.3]. These properties depend on microstructural characteristic, crystal phase, grain morphology and aggregation, and also on crystallite orientation (Chateigner et al. 2000).

The underlying secret of the above phenomena is the organic matrix existing in the mollusk shells. The matrix of shells can be separated into 2 broad classes: the soluble matrix (SM) and the insoluble matrix (IM). Soluble matrix is generally acidic due to high mole fractions of Asp and Glu, which are considered to play important roles in crystal nucleation, crystal growth and inhibition, crystal polymorphism, and atomic lattice orientation (Weiner & Addadi 1991, Wheeler 1992, Falini et al. 1996, Belcher et al. 1996). On the contrary, the insoluble matrix has a relatively high proportion of nonpolar amino acids, which are believed to provide the organic matrix framework for calcium carbonate to deposit (Weiner & Traub 1980, Weiner 1986).

CaC[O.sub.3] has 3 known polymorphs: calcite, aragonite, and vaterite phases. The calcite and aragonite are stable polymorphs and observed in nature, whereas the vaterite is a metastable polymorph and rarely seen in biologic systems (Weiner & Addadi 1997). Addadi & Weiner (1985) demonstrated that the biomacromolecules from shell could exert control over the polymorphism of a particular mineral phase. Previous studies on in vitro crystallization have shown that soluble proteins, associated with a variety of calcium carbonate biominerals, are able to interact with calcite growth and affect a variety of crystal properties (Berman et al. 1988, Berman et al. 1993, Didymus et al. 1993, Wierzbicki et al. 1994, Aizenberg et al. 1994, Sims et al. 1995). Falini et al. (1996) showed that macromolecules extracted from the aragonite layer of mollusk shells induced nucleation of aragonite crystals under an appropriate microenvironment. Moreover, Belcher et al. (1996) demonstrated that a 16-kDa soluble polyanionic protein is sufficient to allow the transformation from the calcite to the aragonite phase. However, the exact mechanisms by which the macromolecules control the polymorphism, in both biotic and abiotic systems, are not yet well understood.

The shells of abalone are, in some cases, exclusively calcitic (e.g., Haliotis kamtschatkana, Haliotis rufescens) or aragonitic (e.g., Haliotis asinina, Haliotis glabra), but in many species the shell contains both calcite and aragonite (e.g., Haliotis tuberculata) (Dauphin et al. 1989). The diversity of crystal compositions in the shell of abalone presents a unique and interesting model for exploring shell biomineralization. As results on the biologic mechanisms of biomineralization have been obtained previously on the abalone Haliotis discus hannai Ino, it can be considered as a model to study the mineralization, using an interdisciplinary approach (Mai et al. 2003).

Until now there remains a large gap between the understanding of chemical model systems and biochemical/physiologic processes that control shell formation. The aim of this study is to investigate the inorganic part of the calcium carbonate shell of H. discus hannai Ino and to begin to elucidate the roles of the soluble matrix from abalone shell on the in vitro crystallization of calcium carbonate.

MATERIALS AND METHODS

Scanning Electron Microscopy Observation of Abalone Shell

Samples were fractured from the growing margin of abalone shell (shell length: 8.5 [+ or -] 0.3 cm, body weight: 75.3 [+ or -] 3.2g) and mounted on a stub, vacuum-coated with gold-palladium for 5 min in a Technics gold-sputter, and scanned using a scanning electron microscopy (scanning electron microscopy, JEOL-840). Different layers of shell were subject to x-ray diffraction on a Rigaku diffracmeter, which used a Cu K[alpha] radiation and operated at 40 KV and 100 mA, set to run from 25[degrees] to 60[degrees] at a step size of 0.01[degrees] 2[theta] and a counting time of 5 sec.

IR Spectrometry Analysis

All spectra were recorded at 4 [cm.sup.-1] resolution with 64 scans with a strong Norton-Beer apodization on a Perkin-Elmer model 1600 Fourier transform IR spectrometer (FTIR), in the wave-number range 4000-600 [cm.sup.-1]. The system was purged and permanently maintained under nitrogen to reduce atmospheric C[O.sub.2] and [H.sub.2]O absorption. A background spectrum was measured for pure KBr. Sample spectra were automatically ratioed against background to minimize C[O.sub.2] and [H.sub.2]O bands. All samples and KBr were dried in an oven at 38[degrees]C overnight and then ground into fine powder in an agate mortar for 10 min.

Isolation of the Soluble Matrix Proteins

The nacreous layer and prismatic shell were separated by mechanical scraping, then were crushed and pulverized into fine powder, respectively. Powders were suspended in 3,500 molecular weight cutoff dialysis membranes (Cole-Parmer Instrument, USA) and dialyzed against 5% acetic acid containing 0.01% (w/v) sodium azide for 72 h at room temperature. This was followed by exhaustive dialysis against ultrapure water for 48 h to remove the acetic acid. The resultant dialysate was centrifuged at x20,000g for 30 min at 4[degrees]C, which separated the supernatant (SM) and precipitated (IM) fractions. The supernatant was collected and lyophilized for subsequent experiments.

Crystal Growth Experiments

CaC[O.sub.3] crystals were grown on glass coverslips in a Ca[Cl.sub.2] solution. Briefly, 3 mL of 12 mM Ca[Cl.sub.2] solution was introduced into the wells containing the coverslips. The wells were covered with aluminum foil with punctures and placed inside a closed desiccator for 2 days for crystal growth by the slow diffusion of gases released by the decomposition of ammonium carbonate placed inside the desiccator (Rajamani et al. 2003). To study the role of soluble matrixes from nacreous and prismatic shells on CaC[O.sub.3] crystallization, aliquots of protein (10 [micro]g/mL) were introduced into each well. After 2 days, the coverslips were carefully lifted from the crystallization vessels, rinsed gently with Millipore water, and air dried at room temperature for further analysis.

Characterization of Synthesized Crystals

The morphologies of the crystals were observed under an Olympus BH-2 phase contrast microscope (Olympus) with a digital camera (Pixera, Pro 150ES). The crystals were then scraped and characterized by FTIR.

RESULTS

Shell Structure of Abalone Shell

There are 3 layers sequentially from the exterior to the interior of the abalone shell (Fig. 1). An uncalcifled layer, called the periostracum, covers the shell externally. The calcified layer is composed of 2 different polymorphs of calcium carbonate: calcite and aragonite. The majority of the calcite forms the prismatic layer of the shell. The aragonite, formed between the mantle and the prismatic layer, is deposited as rounded or polygonal oriented (001) tablets. These tablets are ~0.2-0.3 [micro]m thick and ~2-10 [micro]m wide. The tablets are arranged in columns to form a stack with their c-axis aligned in the growing margin.

FTIR Analysis of Prismatic Powder and Nacre Powder

Figure 2 shows that both prismatic and nacreous powders were composed of organic matrix and mineral phase with numerous bands from 4000 [cm.sup.-1] to 600 [cm.sup.-1]. The spectra of calcite (prismatic powder) are characterized by 3 prominent absorption maxima at 1430-1450 [cm.sup.-1] (v3), 876 [cm.sup.-1] (v2) and 710 [cm.sup.-1] (v4), and one minor band at 1012-1087 [cm.sup.-1] (v1). The splitting of v4, which is prevalent in the spectra of aragonite, is absent from calcite. The IR spectra of nacreous powder are characterized by 3 prominent absorption maxima at 1470-1490 [cm.sup.-1] (v3), 858 [cm.sup.-1] (v2), and 712-700 [cm.sup.-1] (v4). Bands of lesser intensity appear at 1785 [cm.sup.-1] and 1060-1080 [cm.sup.-1] (v1). Another absorption area in the range of 2520-2650 [cm.sup.-1] exists in both the nacreous and prismatic powders.

In vitro Crystallization of Calcium Carbonate

Figure 3 shows the morphologies and FTIR spectra of the crystals grown on the coverslips with or without soluble matrix from different origins. Figure 3a shows the morphology of crystals without matrix protein supplementation. Rhombohedral calcite crystals were observed in the absence of matrix protein.

Figure 3b shows the morphology of crystals grown in the solution with prismatic soluble proteins and its FTIR spectra. The family of proteins from the prismatic layer induced a habit modification in calcite growth, yielding globular growth instead of the rhombohedra obtained in the absence of proteins. This may be the result of their relatively different rates of growth in the various directions. In the control treatment (Fig. 3a), calcite crystals show fuzzy step edges and the corners between step edges are sharp, whereas in the calcite grown in the presence of prismatic soluble matrix, step edges appear highlighted and corners between edges have become round.

In the nacreous soluble matrix supplemented solution, only aragonites are deposited, identified by the splitting of v4 in the FTIR spectra of deposited crystals as shown in Figure 3c. Hedgehog-like aggregates, with outward-oriented needles and their agglomerates, are formed.

DISCUSSION

In the present study, FTIR spectra showed an absorption area in the range of 2520-2650 [cm.sup.-1] in both the nacreous and prismatic layer powder of Haliotis discus hannai, indicating the presence of HC[O.sub.3.sup.-]. FTIR analysis of the nacre from Pinctada maxima also found HC[O.sub.3.sup.-] group and demonstrated that HC[O.sub.3.sup.-] group participated in the formation of calcium carbonate (Balmain et al. 1999). Studies have shown that nacrein, a 60 kDa protein isolated from the EDTA-soluble organic matrix of both nacreous and prismatic layers of Pinctada maxima, exhibits carbonic anhydrase activity (CA), suggesting that carbonic anhydrase acts to catalyze HC[O.sub.3.sup.-] to carbonate ions at the site of crystal growth, and that this is important for the formation of both aragonite and calcite (Miyamoto et al. 1996, Miyashita et al. 2002).

In the in vitro crystallization experiment, the soluble matrix from the nacreous layer induced the formation of aragonite, whereas the soluble matrix from the prismatic layer induced the deposition of calcite. It can be presumed that the selection of crystal polymorph, by cooperative interaction with the soluble matrix, showed perfect arrangement with the polymorph of calcium carbonate from which the soluble matrix had been isolated, suggesting that the polymorph selection of crystal is mediated by the differential production of these two different families of soluble matrix. The present results, and previous findings in other mollusk species (Belcher et al. 1996, Falini et al. 1996), have demonstrated that soluble matrix proteins, rather than insoluble ones, can control crystal polymorphism in vitro.

The deposition of less stable aragonite crystals was induced by the soluble matrix from the nacreous layer. Meanwhile, the screw character of calcite was deposited in the prismatic soluble matrix supplemented solution. These results indicate a strong interaction between the acidic groups present in the proteins and the growing crystals. It has been proposed that the interaction of proteins with specific crystal planes is governed by intrinsic stereochemical recognition of the macromolecules for specific molecular motifs exposed on certain crystal planes, which implies the stimulation of crystal formation at certain sites and relatively inhibition of the process at all other sites (Weiner & Addadi 1991, Mann et al. 1993). It is suggested that the nature and organization of functional groups at the surface of the proteins are crucial to achieve the desired selectivity in polymorph nucleation, as well as controlling the crystalline nature and morphology of the inorganic phase.

The weakness of the present study is that unpurified proteins have been applied in the in vitro mineralization experiments. It remains to determine which, if any, of the polyanionic proteins become occluded within the growing crystals, and with which crystallographic faces the individual proteins interact, To understand the underlying mechanisms of the interactions between proteins and mineral, it is essential to know the primary structure of the proteins involved. As a systematic work, we have purified the most dominate soluble protein in the nacre layer to homogeneity and a molecular approach is underway (unpublished data).

ACKNOWLEDGMENTS

This study was financially supported by grant No. 2001AA628080 and No.2004AA628100 from the National High Technology Research and Development Program of China (863 Program) and grant No. 30200215 from the National Natural Science Foundation of China (NNSFC).

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JIANMIN ZHAO, WENBING ZHANG, KANGSEN MAI, * WEI XU, ZHIGUO LIUFU, HONGMING MA, QINGHUI AI, AND BEIPING TAN

The Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, People's Republic of China

* Corresponding author. E-mail: [e-mail]