410
the oxidation of capsular catechols to highly reactive quinones; forming dark
pigments which tan the capsular matrix.
One of limiting factors that hindered detailed characterization of the proteins that
comprise elasmobranch egg capsules is their strong resistance to acidic treatment.
Only hot alkali and strong mineral acids can dissolve the tanned capsule (Hussakof
and Welker 1911 ). Acid hydrolysis of the whole capsular material led to obtaining
of the hydrolisate where elevated glycine, proline and hydroxyproline have been
identifi ed as dominant amino acids (Krishnan 1959 ; Gross et al. 1958 ; Knight and
Hunt 1974 ; Cox et al. 1987 ). Thus, the collagenous origin of this biological material
has been suggested. However, levels of glycine, proline and hydroxyproline in the
capsules are generally lower than those of typical collagens. Identifi cation of high
levels of tyrosine in corresponding extracts of these capsules, suggesting the
presence of another protein rich in tyrosine (Gross et al. 1958 ; Knight and Hunt
1974 ). Six proteins ranging in molecular mass from 100 to 15 kDa comprise the
bulk of the skate egg capsule material (Koob and Cox 1993 ). In the little skate,
Leucoraja erinacea , none of these proteins have an amino acid composition
observed in interstitial collagens.
Nonetheless, glycine accounts for a relatively large proportion in each protein
(50 % in the two low molecular mass proteins), and the larger molecular mass proteins contain substantial amounts of proline and hydroxyproline, suggesting that
one or more of these proteins may contain collagenous domains. Of principal relevance to the polymerization mechanism described below, these proteins contain a
relative abundance of tyrosine (25 % in the two low molecular mass proteins) (Koob
2002 ). Thus, skate egg capsules are polymerized by a quinone tanning process
involving the introduction and oxidation of ortho-catechols following secretion and
assembly of capsule precursors in the shell gland (Koob and Cox 1990 ). Catechol
formation occurs via hydroxylation of tyrosine residues in the structural protein
phase of the material. Approximately 50 % of the overall tyrosine residues are
catalytically hydroxylated, forming peptide-bound 3,4-dihydroxyphenylalanine
(an ortho-catechol).
As reported above, Catechol oxidase catalyzed oxidation of Dopa residues
follows, producing Dopa quinone (Koob and Cox 1988 ). Dopa quinone then polymerizes the matrix through an as yet uncharacterized condensation reaction. Based on
evidence from experiments with Dopa containing synthetic peptides, it seems likely
that Dopa-quinones on neighboring protein monomers produce covalent cross-links
via bisquinone formation (Koob 2002 ).
The resulting quinone-tanned material is mechanically and chemically stable.
The polymerized material can be solubilized only with hydrolytic agents such as
strong acids and bases (e.g. 6 N HCl at 100 °C and boiling 5 % KOH). It is refractory to proteinase attack, chaotropic agents, and organic solvents. As proposed
by Thomas Koob, three aspects of this biological material elicit interest from a
biomimetics perspective.
First, the polymerization that occurs after secretion and assembly of the precursors is
very rapid. Capsule material in the little skate undergoes a transition from a chemically
and mechanically friable substance to a leather-like material in less than 12 h, and
12 Egg-Capsule Proteins of Selachians
the oxidation of capsular catechols to highly reactive quinones; forming dark
pigments which tan the capsular matrix.
One of limiting factors that hindered detailed characterization of the proteins that
comprise elasmobranch egg capsules is their strong resistance to acidic treatment.
Only hot alkali and strong mineral acids can dissolve the tanned capsule (Hussakof
and Welker 1911 ). Acid hydrolysis of the whole capsular material led to obtaining
of the hydrolisate where elevated glycine, proline and hydroxyproline have been
identifi ed as dominant amino acids (Krishnan 1959 ; Gross et al. 1958 ; Knight and
Hunt 1974 ; Cox et al. 1987 ). Thus, the collagenous origin of this biological material
has been suggested. However, levels of glycine, proline and hydroxyproline in the
capsules are generally lower than those of typical collagens. Identifi cation of high
levels of tyrosine in corresponding extracts of these capsules, suggesting the
presence of another protein rich in tyrosine (Gross et al. 1958 ; Knight and Hunt
1974 ). Six proteins ranging in molecular mass from 100 to 15 kDa comprise the
bulk of the skate egg capsule material (Koob and Cox 1993 ). In the little skate,
Leucoraja erinacea , none of these proteins have an amino acid composition
observed in interstitial collagens.
Nonetheless, glycine accounts for a relatively large proportion in each protein
(50 % in the two low molecular mass proteins), and the larger molecular mass proteins contain substantial amounts of proline and hydroxyproline, suggesting that
one or more of these proteins may contain collagenous domains. Of principal relevance to the polymerization mechanism described below, these proteins contain a
relative abundance of tyrosine (25 % in the two low molecular mass proteins) (Koob
2002 ). Thus, skate egg capsules are polymerized by a quinone tanning process
involving the introduction and oxidation of ortho-catechols following secretion and
assembly of capsule precursors in the shell gland (Koob and Cox 1990 ). Catechol
formation occurs via hydroxylation of tyrosine residues in the structural protein
phase of the material. Approximately 50 % of the overall tyrosine residues are
catalytically hydroxylated, forming peptide-bound 3,4-dihydroxyphenylalanine
(an ortho-catechol).
As reported above, Catechol oxidase catalyzed oxidation of Dopa residues
follows, producing Dopa quinone (Koob and Cox 1988 ). Dopa quinone then polymerizes the matrix through an as yet uncharacterized condensation reaction. Based on
evidence from experiments with Dopa containing synthetic peptides, it seems likely
that Dopa-quinones on neighboring protein monomers produce covalent cross-links
via bisquinone formation (Koob 2002 ).
The resulting quinone-tanned material is mechanically and chemically stable.
The polymerized material can be solubilized only with hydrolytic agents such as
strong acids and bases (e.g. 6 N HCl at 100 °C and boiling 5 % KOH). It is refractory to proteinase attack, chaotropic agents, and organic solvents. As proposed
by Thomas Koob, three aspects of this biological material elicit interest from a
biomimetics perspective.
First, the polymerization that occurs after secretion and assembly of the precursors is
very rapid. Capsule material in the little skate undergoes a transition from a chemically
and mechanically friable substance to a leather-like material in less than 12 h, and
12 Egg-Capsule Proteins of Selachians