also be used to describe the cooperativity of the
12-mer from Homarus americanus and of the 48mer of Limulus polyphemus. Cooperative basic
units present in both 6-mers and 12-mers may
each be in either the T or the R state [25, 40].
Homohexamers can arise from certain haemocyanin subunits of various crustaceans and chelicerates; these may even show weak cooperativity, e.g.
the subunit Lim-II with n = 1.8 [108, 111].
The importance of subunit interaction for the
respiratory properties of the whole molecule can
be examined by the use of toxic mercury salts.
Each Eurypelma subunit binds 1-2 Hi+; 24mers formed from reaggregated subunits treated
in this way have the same binding characteristics
as isolated subunits, i.e. all interaction is blocked.
The use of individually poisoned subunits in the
reaggregation of the 24-mers of Eurypelma or of
the 48-mers of Limulus has facilitated the study
of their roles in the complete molecule [108, 115].
The O2 affinity of haemocyanins from many crustaceans is increased by L-Iactate. D-Iactate, glycolate and pyruvate are less active and D- and Lalanine have no effect; thus, all four substituents
of the asymmetric carbon atom are involved in
the interaction. The strength of the reaction
varies with the species; the value of L\logp501
L\[L-Iactate] varies from -0.096 (Carcinus maenas) to -0.560 (Palaemon elegans). The strength
of the Bohr effect, with (Maja squinado) and -1.40 (Liocarcinus depurator), is not correlated to the lactate effect [108].
The haemolymph contains other unknown haemocyanin modulators (UF, unidentified factors)
in addition to lactate; a part of the UF activity can
be ascribed to uric acid [94, 108].
7.3.2 Haemocyanins of the Molluscs
The edible snail Helix pomatia possesses three
different haemocyanin forms to which the names
a-Hc, ~s-Hc and ~c-Hc were initially given. The aHc, which makes up 75 % of the total, is characterized by the fact that it dissociates into two halves in 1 mol/1 NaCI at pH 5.7. Of the nondissociated ~-Hc, one part (30-50 %) precipitates
at pH 5.3 (~c-Hc) and the rest remains in solution
(~s-Hc). After it was discovered that a-Hc and ~sHc are made up from the same subunits (a and
a'), the haemocyanins were renamed aD-Hc (dissociating) and aN-Hc (non-dissociating); the
name ~c-Hc for the third component, which is
made up of different (type ~) subunits, was
retained. ~c-Hc is the most investigated form. The
7.3.2 Haemocyanins of the Molluscs
279
haeniocyanin of Helix aspersa may also be
divided into three fractions [52]. In electron micrographs, the baemocyanin molecules from Helix
pomatia and other gastropods appear as hollow
cylinders that are 35 nm in diameter and 38 nm
long; the ends are partly closed by two so-called
collars (Fig. 7.11a). This haemocyanin sediments
in the ultracentrifuge with 100S, corresponding to
9 MDa, but molecules of 120S, i.e. with 1.5 times
greater mass, are also found. At weakly alkaline
pH and low Ca2+ concentration, gastropod haemocyanin molecules split into fragments of 60S
(half),20S (1/10) and 11S (1120). Each subunit (II
20) consists of eight domains, each with one Or
binding site, and appears in the electron microscope as a chain of eight spheres with a diameter
of 5.5 nm. The copper content of about 0.23 %
gives a mass of 55 kDa per 02-binding site. In
marine snails of the family Melongenidae, which
includes the genera Melongena and Busycon, the
haemocyanins exist as extended, tubular aggregates of five or more 60S half molecules; they are
consistently organized such that both ends of the
tubulus are closed by a collar [63]. Partial tryptic
digest of the subunits gives eight fragments (a-h)
with a mass of 55 kDa, six of which form the wall
of the tubulus and two form the collar. In the 1/10
fragments, the two subunits are apparently
arranged in parallel (Fig. 7.11a). The eight fragments of the subunits differ greatly in their amino
acid and carbohydrate composition, and in their
functional properties. For example, the carbohydrate content in Helix pomatia varies from
17.6 % for fragment g to 0.7% for fragment c,
with a mean of 8.2 % [108, 188]. The structures of
the carbohydrates have been analysed in H. pomatia and Lymnaea stagnalis (Fig.7.10b). They
differ from the N-linked oligosaccharides of other
animals by the presence of xylose and 0methylated sugars. The association of further
sugars can lead to their transformation into complex carbohydrates of greater molecular mass
[93]. The methylated sugars are not incorporated
as such, but are later methylated with adenosylmethionine as the cofactor.
The functional subunit d from the ~c-Hc of
H. pomatia has been completely sequenced. The
sequence of 410 amino acids agrees 42 % with the
399-amino-acid C-terminal domain of octopus
Hc, which was sequenced via the cDNA [96];
however, it exhibits homology to arthropod haemocyanins only in a small, 42-amino-acid fragment. This segment corresponds to the Cu(B)
region of the haemocyanin from the spiny lobster
Panulirus interruptus,. homologous sequences are
12-mer from Homarus americanus and of the 48mer of Limulus polyphemus. Cooperative basic
units present in both 6-mers and 12-mers may
each be in either the T or the R state [25, 40].
Homohexamers can arise from certain haemocyanin subunits of various crustaceans and chelicerates; these may even show weak cooperativity, e.g.
the subunit Lim-II with n = 1.8 [108, 111].
The importance of subunit interaction for the
respiratory properties of the whole molecule can
be examined by the use of toxic mercury salts.
Each Eurypelma subunit binds 1-2 Hi+; 24mers formed from reaggregated subunits treated
in this way have the same binding characteristics
as isolated subunits, i.e. all interaction is blocked.
The use of individually poisoned subunits in the
reaggregation of the 24-mers of Eurypelma or of
the 48-mers of Limulus has facilitated the study
of their roles in the complete molecule [108, 115].
The O2 affinity of haemocyanins from many crustaceans is increased by L-Iactate. D-Iactate, glycolate and pyruvate are less active and D- and Lalanine have no effect; thus, all four substituents
of the asymmetric carbon atom are involved in
the interaction. The strength of the reaction
varies with the species; the value of L\logp501
L\[L-Iactate] varies from -0.096 (Carcinus maenas) to -0.560 (Palaemon elegans). The strength
of the Bohr effect, with (Maja squinado) and -1.40 (Liocarcinus depurator), is not correlated to the lactate effect [108].
The haemolymph contains other unknown haemocyanin modulators (UF, unidentified factors)
in addition to lactate; a part of the UF activity can
be ascribed to uric acid [94, 108].
7.3.2 Haemocyanins of the Molluscs
The edible snail Helix pomatia possesses three
different haemocyanin forms to which the names
a-Hc, ~s-Hc and ~c-Hc were initially given. The aHc, which makes up 75 % of the total, is characterized by the fact that it dissociates into two halves in 1 mol/1 NaCI at pH 5.7. Of the nondissociated ~-Hc, one part (30-50 %) precipitates
at pH 5.3 (~c-Hc) and the rest remains in solution
(~s-Hc). After it was discovered that a-Hc and ~sHc are made up from the same subunits (a and
a'), the haemocyanins were renamed aD-Hc (dissociating) and aN-Hc (non-dissociating); the
name ~c-Hc for the third component, which is
made up of different (type ~) subunits, was
retained. ~c-Hc is the most investigated form. The
7.3.2 Haemocyanins of the Molluscs
279
haeniocyanin of Helix aspersa may also be
divided into three fractions [52]. In electron micrographs, the baemocyanin molecules from Helix
pomatia and other gastropods appear as hollow
cylinders that are 35 nm in diameter and 38 nm
long; the ends are partly closed by two so-called
collars (Fig. 7.11a). This haemocyanin sediments
in the ultracentrifuge with 100S, corresponding to
9 MDa, but molecules of 120S, i.e. with 1.5 times
greater mass, are also found. At weakly alkaline
pH and low Ca2+ concentration, gastropod haemocyanin molecules split into fragments of 60S
(half),20S (1/10) and 11S (1120). Each subunit (II
20) consists of eight domains, each with one Or
binding site, and appears in the electron microscope as a chain of eight spheres with a diameter
of 5.5 nm. The copper content of about 0.23 %
gives a mass of 55 kDa per 02-binding site. In
marine snails of the family Melongenidae, which
includes the genera Melongena and Busycon, the
haemocyanins exist as extended, tubular aggregates of five or more 60S half molecules; they are
consistently organized such that both ends of the
tubulus are closed by a collar [63]. Partial tryptic
digest of the subunits gives eight fragments (a-h)
with a mass of 55 kDa, six of which form the wall
of the tubulus and two form the collar. In the 1/10
fragments, the two subunits are apparently
arranged in parallel (Fig. 7.11a). The eight fragments of the subunits differ greatly in their amino
acid and carbohydrate composition, and in their
functional properties. For example, the carbohydrate content in Helix pomatia varies from
17.6 % for fragment g to 0.7% for fragment c,
with a mean of 8.2 % [108, 188]. The structures of
the carbohydrates have been analysed in H. pomatia and Lymnaea stagnalis (Fig.7.10b). They
differ from the N-linked oligosaccharides of other
animals by the presence of xylose and 0methylated sugars. The association of further
sugars can lead to their transformation into complex carbohydrates of greater molecular mass
[93]. The methylated sugars are not incorporated
as such, but are later methylated with adenosylmethionine as the cofactor.
The functional subunit d from the ~c-Hc of
H. pomatia has been completely sequenced. The
sequence of 410 amino acids agrees 42 % with the
399-amino-acid C-terminal domain of octopus
Hc, which was sequenced via the cDNA [96];
however, it exhibits homology to arthropod haemocyanins only in a small, 42-amino-acid fragment. This segment corresponds to the Cu(B)
region of the haemocyanin from the spiny lobster
Panulirus interruptus,. homologous sequences are