An important part of this thesis was focused on the search, purification and characterization of the intracrystalline proteins, a new group of proteins embedded in single nacre aragonite platelets.
By using biochemical techniques and small angle neutron scattering, it could be clearly shown for the first time that proteins are included in the aragonite platelets of nacre. With small angle neutron scattering the size of the intracrystalline proteins was determined to be 40 Å and their content at 3-4% of the platelet volume.
The best way to purify these proteins turned out to be the methanol – chloroform precipitation after dissolution of purified aragonite platelets. Three main intracrystalline proteins with a molecular weight of 6, 14 and 25 kDa were obtained.
Unfortunately a further isolation of this three-protein mixture was not achieved. The behaviour of the intracrystalline proteins during gel electrophoresis and ion exchange chromatography suggested that they are possibly negatively charged. The possibly acidic nature of the proteins and the purification difficulties were also reported by other groups [Fu et al., 2005; Gotliv et al., 2003].
The investigation of the influence of the intracrystalline proteins on a growing calcite surface with atomic force microscopy revealed a strong surface modifying effect of these proteins. After the incubation of the proteins on the calcite surface it could be shown that a polymorph transition to aragonite had taken place. In contrast to that, the intracrystalline proteins showed an inhibitory effect on crystallization, when added to a supersaturated calcium carbonate solution.
It can be stated that the interaction of the intracrystalline proteins with calcium carbonate is very strong, possibly depending to their proposed acidic nature. The reason for the different behaviour of the proteins, in a supersaturated solution and on a crystal surface is unclear.
5.1.2 Perlucin
A new assay for the extraction of the nacre water-soluble protein perlucin was developed. A ball mill to prepare nacre for protein extraction was used for the first time. By using this method almost pure perlucin in its native state could be obtained.
The reason for this is unclear; a possible explanation is that during ball milling other nacre proteins get degraded, maybe due to lower temperature stability.
By cocrystallization of calcium carbonate with native perlucin, crystals characterized by a stepped morphology due to the nucleation of new layers were obtained. All newly nucleated crystal faces presented a parallel orientation to the faces beneath.
This result was in accordance with previous investigations that showed that perlucin is able to nucleate flat mineral layers on a calcite surface [Treccani et al., 2003; Blank et al., 2003].
5.1.3 Perlinhibin
It could been shown that perlinhibin, a small water-soluble protein, is an inhibitor calcium carbonate crystal growth. When incubated on a calcite surface with a supersaturated calcium carbonate solution perlinibin adheres to mineral step edges and inhibits the growth of new mineral at the binding site. Further more it could be shown that perlinhibin has the capability to induce aragonite formation. The complementary ability of perlinhibin to suppress the growth of calcite and induce the formation of aragonite suggests that this small protein is maybe involved in the highly controlled polymorph regulation.
5.1.4 Poly-γ-methyl-L-glutamate
Crystallization of calcium carbonate with poly-γ-methyl-L-glutamate induces the precipitation of flat crystals, with a shape similar to that of natural aragonite platelets.
The crystals precipitated with the vapour diffusion techniques presented a stepped morphology, also found in the crystals precipitated with perlucin at very low concentrations. The common features between the calcium carbonate crystals precipitated in presence of poly-γ-methyl-L-glutamate and perlucin may be an indication of some analogies between the molecular structure of poly-γ-methyl-L -glutamate and perlucin.
Because poly-γ-methyl-L-glutamate seems to interact with calcium carbonate in a way similar to that of nacre proteins, poly-γ-methyl-L-glutamate might provide a valid candidate to model the interactions taking place between native nacre molecules and
atomic scale.
5.2 Perspectives
The intracrystalline proteins in nacre of H. laevigata need further investigation. Each single component has to be isolated and its structure as function as well should be characterized. This may lead to a more precise and detailed understanding of the protein-mineral interactions.
Besides the characterization of intracrystalline proteins, other water-soluble proteins have to be investigated. A wider amount of information about the protein structure may lead to a better understanding of their role in nacre formation. Because the formation of nacre is a result of the cooperative interaction of all its organic molecules and proteins, more information about the interplay – spatial and temporal - of the different organic components has to be obtained.
In addition to that, the interactions of the soluble organic - matrix components with the insoluble matrix, especially with chitin should be elucidated. This is of special importance because recent investigations from our lab showed that the water-insoluble matrix induces in vitro the formation of stacks of aragonite platelets [Heinemann et al., 2006]. It seems likely that soluble matrix components are partially bound to the water - insoluble chitin matrix during growth of nacre, maybe working as crystallization nucleators when bound to a surface and inhibitors, when dissolved.
Besides the clarification of the processes involved in the nacre formation in vivo, synthetic molecules which template or influence mineral formation in a desired way should be found. This may lead the way to novel composite materials with tuneable properties. On the other side the investigation of such synthetic molecules in calcium carbonate mineralization may also help to understand the principles of biomineralization.