Discussion

The biochemical characterization of the intracrystalline proteins was a very challenging and fundamental step for the investigation of nacre intracrystalline proteins. Numerous technical problems related to the nature of the proteins were encountered and hindered the investigation. The consolidated biochemical analysis used for the purification of the other nacre proteins, as perlucin and perlinhibin, resulted to be inappropriate for the intracrystalline proteins, therefore it was necessary to develop new strategies.

By demineralization of aragonite platelets with EDTA a higher amount of proteins was obtained compared with that of acetic acid demineralization. The amount of

Demineralization of nacre with acetic acid led to a low amount of proteins. Maybe intracrystalline proteins are sensitive and at the low pH of acetic acid solutions (pH <

2.2) they might undergo hydrolysis.

The higher amount of proteins obtained by EDTA demineralization was maybe a consequence of the conditions at which demineralization was carried out (pH neutral). Under these conditions denaturation of the proteins may be hindered.

The flakes found after complete demineralization of the mineral phase could presumably be protein aggregates, denaturated proteins, chitin or protein-chitin complexes. Chitin remnants could be still present between some platelets and can released only after demineralization. The nature of these flakes is still under investigation.

Three main intracrystalline proteins with an approximate molecular weight of 6, 15 and 25 kDa were detected. Other proteins with a size between 3.5 and 6 kDa were detected and typically appeared as diffused bands in electrophoresis gel slabs. The nature of these small proteins is not well characterized, they could be peptides or protein fragments formed after unknown degradation processes.

Precipitation with ammonium sulfate revealed to be not suitable to precipitate intracrystalline proteins. This is maybe due to the incapability, in some cases, of ammonium sulfate to bind to Ca²⁺-binding proteins [Lottspeich and Zorbas, 1998], or proteins diffused out of the gel during electrophoresis, as reported in [Gotliv et al., 2003].

After gel electrophoresis of proteins precipitated with trichloroacetic acid, proteins did not appeared as bands but diffused and smeared. It was observed that the time between protein extraction and analysis was a determining factor influencing the protein concentration. This may indicate that intracrystalline proteins might easily degrade after their extraction from the mineral phase, although during the investigations constant storing conditions were carefully maintained and monitored.

Interesting was the spontaneous formation of crystals after precipitation with trichloroacetic acid. XRD analysis did not led to recognition of any known mineral and left several questions open. The crystals could not be a calcium carbonate polymorph, formed after recrystallization of the dissolved mineral; neither the crystal was calcium acetate that could eventually be formed by the presence of calcium ions

and acetic acid in the solution. One possibility is that the crystals are protein-mineral crystals. Unfortunately no clear results are available.

Methanol-chloroform precipitation revealed to be the best method to precipitate proteins and to get mostly clear data about their molecular weight. Gel electrophoresis clearly showed the existence of three proteins with an approximate molecular weight of 6, 14 and 25 kDa. Unfortunately more precise information about their molecular weight by mass spectroscopy could not be obtained.

MALDI analysis and data base search did not lead to identification of known proteins.

A large number of the peptides obtained showed a weight loss of 64 Dalton, which could be the result of a labile modification of amino acid residues. The difficulties during mass spectroscopy investigation seems to be related to an unknown modification of the protein, maybe induced during protein extraction with organic solvent (personal communication with Dr. Anja Resemann and Dr. Markus Meyer, Bruker Daltonics GmbH, Bremen, Germany).

During the whole investigation an unusual behaviour of the proteins was observed during electrophoresis. Electrophoresis gels typically presented a poor resolution of the protein bands after staining and the band appeared mostly diffused and smeared.

The same phenomenon, also reported in [Gotliv et. al., 2003], could be related to a high charge density of the proteins. Eventually intracrystalline proteins have a high tendency to form agglomerates that cannot easily enter the gel. Furthermore intracrystalline proteins may bind poorly with sodium dodecyl sulfate, an anion detergent used to defold the tertiary structure of proteins. Complete defolding of the proteins is maybe not achieved and proteins could remain partially aggregated. This was suggested by the fact that some of the protein did enter the gels. The same phenomenon was also reported in [Dauphin and Cuif, 1997, Gotliv et al., 2003].

The weak binding of sodium dodecyl sulfate to proteins and their anomalous migration in gels is a phenomenon often detected with highly anionic proteins. This is supposed to depend on the protein charge densities [Dauphin and Cuif, 1997]. The same behaviour is also shown by Glu-rich recombinant proteins [McGrath et al., 1992].

Sometimes no bands at were visible after SDS-PAGE and Coomassie staining, suggesting that intracrystaline proteins show a tendency to diffuse from the gel, this is typical for proteins presenting an acidic nature. Coomassie Blue stain bound poorly

or aromatic residues [Gotliv et al., 2003].

Differential staining also indicated that the intracrystalline proteins should have an acidic nature and present calcium-binding sites. The possible presence of sialo- and phosphoproteins cannot be neglected as suggested in [Kevin et al., 1983, Goldberg et al., 1997]. Analogous results were also observed with other acid nacre proteins [Fu et al., 2005].

The results obtained by chromatographic separation (ion exchange chromatography and HPLC) were in accordance with the data obtained after precipitation techniques.

Gel electrophoresis of the protein fractions collected after chromatography showed the presence of proteins with approximate molecular weights of 6 kDa, 14 kDa and 25 kDa respectively. In particular ion exchange chromatography could be performed using an anion exchanger indicating that a physiological pH-value of most of the intracrystalline proteins presented a dominant negative charge. A cation exchanger, revealed to be inadequate for separation of intracrystalline proteins.

All these observations lead to a first conclusion that the intracrystalline proteins are a novel group of proteins detected in nacre of the H. laevigata. Intracrystalline proteins maybe characterized by a high density charge, high tendency to form agglomerates and an overall acidic nature, furthermore they seemed to be sensitive and degrade easily.