which was confirmed by the heterogeneity of amino acid compositions of isolated
peaks (Marin 1992). Furthermore, Marin (1992) showed that the intraprismatic
matrix was much more acidic than the soluble matrix associated to nacre. However,
none of the fractionation technique used was able to obtain single macromolecular
components that could be sequenced further. The reasons of this failure were that
the macromolecules associated to prisms and nacre – and more generally to all
calcified tissues – are not globular and that many of them are negatively charged.
In addition, they tend to be polydisperse, and are frequently submitted to posttranslational modifications, such as glycosylation or phosphorylation. Consequently,
they behave anomalously during chromatographical fractionation (for technical
informations on acidic proteins, see the review of Marin and Luquet 2007). These
combined technical obstacles precluded for several years the possibility to purify a
single protein and to obtain partial or full amino acid sequences.
13.4.2 Electrophoresis and Serology on the Shell Matrix
of Pinna nobilis
Decisive analytical improvements were brought in biomineralization investigations, by using polyacrylamide gel electrophoresis in denaturing conditions. By
extracting acido-soluble matrices from prism and nacre of Pinna nobilis, Marin
et al. (1994) showed that the two matrices exhibited similarities and differences:
none of them stained accurately with the classical Coomassie Brilliant Blue for the
reasons indicated here above; when stained with silver nitrate, the two electrophoretic profiles were different but both were characterized by few discrete macromolecular components (from 10 to above 50 kDa), embedded in smearing polydisperse
macromolecules; in the prisms matrix, one prominent band was observed around
15 kDa, and another one, at higher molecular weight; the biggest difference was
observed when employing alcian blue for revealing polyanionic substances, such as
sulfated sugars: the soluble matrix associated to nacre did not stain at all, whereas
the one extracted from prisms gave a strong staining. This clearly suggested that the
prism matrix was extremely acidic (“polyanionic”). We correlated this finding with
the high content of sulfur of the prisms, this finding being also correlated to the high
quantity of soluble sulfated polysaccharides of the intraprismatic matrix.
Another approach, which was also very effective and brought a significant
contribution to the analysis of the shell matrices from Pinna nobilis, was serology,
also called serotaxonomy. As described elsewhere (Muyzer et al. 1984; Collins
et al. 1991; Marin et al. 1999), the method exploits the ability of the mammalian
immune system to produce antibodies against foreign macromolecules, e.g.,
macromolecules extracted from shells. Usually, antibodies recognize short domains,
called epitopes (or antigenic determinants), which, in the case of proteins, are not
longer than 5–8 amino acid residues. Collected antibodies can be tested against a
variety of shell matrices, via different techniques (ELISA, dot blot, Western blots,
13 Acidic Shell Proteins of the Mediterranean Fan Mussel Pinna nobilis
369
peaks (Marin 1992). Furthermore, Marin (1992) showed that the intraprismatic
matrix was much more acidic than the soluble matrix associated to nacre. However,
none of the fractionation technique used was able to obtain single macromolecular
components that could be sequenced further. The reasons of this failure were that
the macromolecules associated to prisms and nacre – and more generally to all
calcified tissues – are not globular and that many of them are negatively charged.
In addition, they tend to be polydisperse, and are frequently submitted to posttranslational modifications, such as glycosylation or phosphorylation. Consequently,
they behave anomalously during chromatographical fractionation (for technical
informations on acidic proteins, see the review of Marin and Luquet 2007). These
combined technical obstacles precluded for several years the possibility to purify a
single protein and to obtain partial or full amino acid sequences.
13.4.2 Electrophoresis and Serology on the Shell Matrix
of Pinna nobilis
Decisive analytical improvements were brought in biomineralization investigations, by using polyacrylamide gel electrophoresis in denaturing conditions. By
extracting acido-soluble matrices from prism and nacre of Pinna nobilis, Marin
et al. (1994) showed that the two matrices exhibited similarities and differences:
none of them stained accurately with the classical Coomassie Brilliant Blue for the
reasons indicated here above; when stained with silver nitrate, the two electrophoretic profiles were different but both were characterized by few discrete macromolecular components (from 10 to above 50 kDa), embedded in smearing polydisperse
macromolecules; in the prisms matrix, one prominent band was observed around
15 kDa, and another one, at higher molecular weight; the biggest difference was
observed when employing alcian blue for revealing polyanionic substances, such as
sulfated sugars: the soluble matrix associated to nacre did not stain at all, whereas
the one extracted from prisms gave a strong staining. This clearly suggested that the
prism matrix was extremely acidic (“polyanionic”). We correlated this finding with
the high content of sulfur of the prisms, this finding being also correlated to the high
quantity of soluble sulfated polysaccharides of the intraprismatic matrix.
Another approach, which was also very effective and brought a significant
contribution to the analysis of the shell matrices from Pinna nobilis, was serology,
also called serotaxonomy. As described elsewhere (Muyzer et al. 1984; Collins
et al. 1991; Marin et al. 1999), the method exploits the ability of the mammalian
immune system to produce antibodies against foreign macromolecules, e.g.,
macromolecules extracted from shells. Usually, antibodies recognize short domains,
called epitopes (or antigenic determinants), which, in the case of proteins, are not
longer than 5–8 amino acid residues. Collected antibodies can be tested against a
variety of shell matrices, via different techniques (ELISA, dot blot, Western blots,
13 Acidic Shell Proteins of the Mediterranean Fan Mussel Pinna nobilis
369