and the late sixties, focused on the amino acid compositions, usually obtained after
hydrochloric acid hydrolysis of the matrix (for an old – but rather complete –
review, see Gre ´goire 1972).
The first biochemical characterization of the shell matrix of Pinna sp. was
performed by Wetzel (1900), who clearly established the proteinaceous nature of
“conchiolin,” by detecting glycine, leucine, tyrosine, and arginine residues in
“conchiolin” hydrolyzates from prisms and nacre. Interestingly, he also showed
that the prisms conchiolin, i.e., the substance that composes the interprismatic
walls, was different in its amino acid composition from the nacre conchiolin.
Half a century later, Roche et al. (1951) and Ranson (1952, 1966) observed that
the “conchiolin” of the calcitic prisms of Pinna sp. had a higher content in Tyr and
Gly and lower content in arginine, serine, and glutamic acid residues than the nacre
conchiolin. Akiyama (1966) also observed a high amount of glycine in the prismatic matrix of Pinna attenuata.
Amino acid analyses on prisms and nacre “conchiolin” of the pinnid bivalves
Pinna nobilis and Atrina nigra were performed by Bricteux-Gre ´goire et al. (1968).
For the nacre of P. nobilis, they obtained a composition dominated by alanine
(29%), glycine (21%), aspartic acid (13%), then serine (9%), while, for prisms,
glycine (37%) and aspartic acid (23%) constituted the dominant amino acids. The
proportions of amino acid residues varied in the same proportions for A. nigra.
Today, we know that the “conchiolin” terminology defines a mixture of different
insoluble constituents that include hydrophobic proteins and chitin.
The discovery that molluscan shell matrices comprise also soluble intracrystalline components (Crenshaw 1972) and the emphasis of their role in calcification process (Weiner et Hood 1975) led many investigators in biomineralization
to focus on this previously undetected organic fraction: not only was the soluble
fraction accessible to amino acid analysis but also to aqueous phase fractionation
techniques, such as chromatography or electrophoresis. At that time, the general
perspective was to purify single proteins and obtain sequence information.
Nakahara et al. (1980) performed series of amino acid analyses on prismassociated matrices of Pinna carnea, Atrina pectinata, and pearl oysters. They
distinguished the interprismatic insoluble walls from an organic fraction that they
considered to be intraprismatic. For the first one (P. carnea), they partly confirmed
previous findings: enrichment in glycine and aspartic acid. They however found
that this matrix was also enriched in tyrosine and cysteine residues, the high amount
of tyrosine suggesting that the interprismatic walls were insolubilized by a quinonetanning process. For the intraprismatic matrix, they measured a striking 72% molar
for aspartic acid (62% in the case of A. pectinata), a fact that fully confirmed the
previous finding of Weiner and Hood (1975).
Few years later, Cuif et al. (1987b, 1988b, 1991), Fre ´rotte (1987), Kervadec
(1990), and Marin (1992) analyzed the soluble matrices from prisms and nacre of
Pinna nobilis by gel permeation or ion-exchange chromatography, both fractionation techniques coupled with subsequent amino acid analysis (after 6 N–HCL
hydrolysis and PITC pre-column derivatization). They clearly evidenced that the
two soluble matrices were heterogeneous in their chromatography profiles, a fact
368
F. Marin et al.
hydrochloric acid hydrolysis of the matrix (for an old – but rather complete –
review, see Gre ´goire 1972).
The first biochemical characterization of the shell matrix of Pinna sp. was
performed by Wetzel (1900), who clearly established the proteinaceous nature of
“conchiolin,” by detecting glycine, leucine, tyrosine, and arginine residues in
“conchiolin” hydrolyzates from prisms and nacre. Interestingly, he also showed
that the prisms conchiolin, i.e., the substance that composes the interprismatic
walls, was different in its amino acid composition from the nacre conchiolin.
Half a century later, Roche et al. (1951) and Ranson (1952, 1966) observed that
the “conchiolin” of the calcitic prisms of Pinna sp. had a higher content in Tyr and
Gly and lower content in arginine, serine, and glutamic acid residues than the nacre
conchiolin. Akiyama (1966) also observed a high amount of glycine in the prismatic matrix of Pinna attenuata.
Amino acid analyses on prisms and nacre “conchiolin” of the pinnid bivalves
Pinna nobilis and Atrina nigra were performed by Bricteux-Gre ´goire et al. (1968).
For the nacre of P. nobilis, they obtained a composition dominated by alanine
(29%), glycine (21%), aspartic acid (13%), then serine (9%), while, for prisms,
glycine (37%) and aspartic acid (23%) constituted the dominant amino acids. The
proportions of amino acid residues varied in the same proportions for A. nigra.
Today, we know that the “conchiolin” terminology defines a mixture of different
insoluble constituents that include hydrophobic proteins and chitin.
The discovery that molluscan shell matrices comprise also soluble intracrystalline components (Crenshaw 1972) and the emphasis of their role in calcification process (Weiner et Hood 1975) led many investigators in biomineralization
to focus on this previously undetected organic fraction: not only was the soluble
fraction accessible to amino acid analysis but also to aqueous phase fractionation
techniques, such as chromatography or electrophoresis. At that time, the general
perspective was to purify single proteins and obtain sequence information.
Nakahara et al. (1980) performed series of amino acid analyses on prismassociated matrices of Pinna carnea, Atrina pectinata, and pearl oysters. They
distinguished the interprismatic insoluble walls from an organic fraction that they
considered to be intraprismatic. For the first one (P. carnea), they partly confirmed
previous findings: enrichment in glycine and aspartic acid. They however found
that this matrix was also enriched in tyrosine and cysteine residues, the high amount
of tyrosine suggesting that the interprismatic walls were insolubilized by a quinonetanning process. For the intraprismatic matrix, they measured a striking 72% molar
for aspartic acid (62% in the case of A. pectinata), a fact that fully confirmed the
previous finding of Weiner and Hood (1975).
Few years later, Cuif et al. (1987b, 1988b, 1991), Fre ´rotte (1987), Kervadec
(1990), and Marin (1992) analyzed the soluble matrices from prisms and nacre of
Pinna nobilis by gel permeation or ion-exchange chromatography, both fractionation techniques coupled with subsequent amino acid analysis (after 6 N–HCL
hydrolysis and PITC pre-column derivatization). They clearly evidenced that the
two soluble matrices were heterogeneous in their chromatography profiles, a fact
368
F. Marin et al.