The extracellular haemoglobins of the plan orbids Helisoma, Planorbis and Biomphalaria have
a completely unique structure. They are large
molecules of 1.75-2.25 MDa which appear in
electron micrographs as decagons with a diameter
of 20 nm. SDS or guanidine breaks up the structure into fragments of 350-380 kDa which are
composed of two disulphide-linked subunits of
175-190 kDa per haem, and thus each subunit
must consist of 10-12 haem-bearing domains. On
the basis of electron microscope studies, it was
previously always assumed that there were up to
ten subunits in the molecule [61, 163]. However,
very accurate measurements of the molecular
mass in Helisoma trivolvis give values of
2.25 MDa for the native haemoglobin and
190 kDa for its subunits; the derived model of 12
subunits arranged on a spherical surface would
appear as a decagon in the electron microscope
[108]. The functional interactions between the
subunits and internally between the different
domains have by no means been fully clarified.
The native haemoglobin of Planorbis corneus has
a p50 of 650 Pa, cooperativity (n :::; 3.4), and an
alkaline Bohr effect, but no other heterotropic
interactions. Partial proteolysis with subtilisin
yields fragments with one haem; these can still
bind O2 but are no longer cooperative. Similar
properties were reported for a haemoglobin present together with haemocyanin in the blood of
the marine snail Aplysia california [163]. The
Biomphalaria haemoglobin is unusual in containing 3 % carbohydrate; one of the two oligosaccharides is bound to an asparagine and the other
to threonine or serine [171].
The blood of the marine mussels Cardita
borealis, C. affinis (Carditidae) and Astarte castanea (Astartidae) contains extracellular haemoglobin with a molecular mass as large as 12 MDa.
This is the largest known respiratory protein, larger even than gastropod haemocyanin, which has
a mass of up to 10 MDa. These haemoglobins
appear in electron micrographs as cylinders that
are 20-34 nm in diameter and 36-120 nm long.
Above pH 9 they break up into fragments of 1.4
MDa; subunits obtained by the usual methods are
about 300 kDA in size. From the haem content it
may be calculated that each subunit carries 1820 haem groups. As in the previously mentioned
case, fragments formed by partial proteolysis contain one haem and bind O2 but show as little
cooperativity as the native haemoglobin [174].
Mussels of the genera Scapharca and Anadara
from the family Arcidae contain two types of haemoglobin in nucleated erythrocytes, homodi7.2.2 Haemoglobins of the Molluscs
271
meric Hb I and tetrameric Hb II, the latter being
made up from two heterodimers. On deoxygenation, Hb II tends to form aggregates of up to four
tetramers. The amino acid sequences of various
Hb I and Hb II chains are now known. All these
chains carry a further helix in front of helix A;
this is known as "N" or "pre-A" and lies parallel
to G and H between H and the EF edge. The
sequences of the haem-binding helices E and F
deviate markedly from those of other haemoglobins; however, the non-polar nature of the haem
pocket is preserved [108, 134, 163]. The sequence
difference between the two haemoglobin types is
greater than the species-specific differences. For
example, the Hb I chains of Anadara broughtoni
and A. trapezia agree at 82 % of positions, but
Hb I and Hb IIa of A. broughtoni are only 45 %
similar [108]. All these mussel haemoglobins are
rather unusually dissociation resistant: 1 m01l1
solutions of sodium iodide, propylurea or guanidine hydrochloride, which dissociate not only
human Hb A but also the more resistant fish haemoglobin, have no effect [17].
The subunits of the homodimer Hb I are
bound via contacts between the E and F helices;
the tetrameric Hb II is composed of two similarly
linked dimers. Whereas helices E and F point outwards in vertebrate haemoglobin dimers, they lie
internally in the haemoglobins of the Arcidae and
are responsible for the stability of the molecule
and for interaction between the subunits [108].
All these haemoglobins have relatively low O2
affinities, equally low Bohr effects and show no
further heterotropic interactions. They are
cooperative, with n values of 1.5 for the dimers
and 2.1 for the tetrameric forms. The responsible
homotropic interactions, however, are fundamentally different from those of vertebrate haemoglobins. In the latter, cooperativity and heterotropic modulation involve interaction between
heterodimers; in the Arcidae, the dimers themselves are already cooperative. Furthermore, vertebrate haemoglobins, with values of n :::; 3.0,
have more effective interaction than mussel haemoglobins [17, 37, 141].
The erythrocytes of other Arcidae, e.g. Noetia
ponderosa or Barbatia virescens, possess only
heterodimeric haemoglobins with subunits of
16 kDa. In addition to such heterodimers, the
related species Barbatia reeveana and B. lima also
contain polymeric haemoglobins of 430 kDa,
consisting of 12-14 subunits of 32 kDa with two
haem-bearing domains. Both domains apparently
arose by gene duplication and consequently show
high sequence similarity with each other and also
a completely unique structure. They are large
molecules of 1.75-2.25 MDa which appear in
electron micrographs as decagons with a diameter
of 20 nm. SDS or guanidine breaks up the structure into fragments of 350-380 kDa which are
composed of two disulphide-linked subunits of
175-190 kDa per haem, and thus each subunit
must consist of 10-12 haem-bearing domains. On
the basis of electron microscope studies, it was
previously always assumed that there were up to
ten subunits in the molecule [61, 163]. However,
very accurate measurements of the molecular
mass in Helisoma trivolvis give values of
2.25 MDa for the native haemoglobin and
190 kDa for its subunits; the derived model of 12
subunits arranged on a spherical surface would
appear as a decagon in the electron microscope
[108]. The functional interactions between the
subunits and internally between the different
domains have by no means been fully clarified.
The native haemoglobin of Planorbis corneus has
a p50 of 650 Pa, cooperativity (n :::; 3.4), and an
alkaline Bohr effect, but no other heterotropic
interactions. Partial proteolysis with subtilisin
yields fragments with one haem; these can still
bind O2 but are no longer cooperative. Similar
properties were reported for a haemoglobin present together with haemocyanin in the blood of
the marine snail Aplysia california [163]. The
Biomphalaria haemoglobin is unusual in containing 3 % carbohydrate; one of the two oligosaccharides is bound to an asparagine and the other
to threonine or serine [171].
The blood of the marine mussels Cardita
borealis, C. affinis (Carditidae) and Astarte castanea (Astartidae) contains extracellular haemoglobin with a molecular mass as large as 12 MDa.
This is the largest known respiratory protein, larger even than gastropod haemocyanin, which has
a mass of up to 10 MDa. These haemoglobins
appear in electron micrographs as cylinders that
are 20-34 nm in diameter and 36-120 nm long.
Above pH 9 they break up into fragments of 1.4
MDa; subunits obtained by the usual methods are
about 300 kDA in size. From the haem content it
may be calculated that each subunit carries 1820 haem groups. As in the previously mentioned
case, fragments formed by partial proteolysis contain one haem and bind O2 but show as little
cooperativity as the native haemoglobin [174].
Mussels of the genera Scapharca and Anadara
from the family Arcidae contain two types of haemoglobin in nucleated erythrocytes, homodi7.2.2 Haemoglobins of the Molluscs
271
meric Hb I and tetrameric Hb II, the latter being
made up from two heterodimers. On deoxygenation, Hb II tends to form aggregates of up to four
tetramers. The amino acid sequences of various
Hb I and Hb II chains are now known. All these
chains carry a further helix in front of helix A;
this is known as "N" or "pre-A" and lies parallel
to G and H between H and the EF edge. The
sequences of the haem-binding helices E and F
deviate markedly from those of other haemoglobins; however, the non-polar nature of the haem
pocket is preserved [108, 134, 163]. The sequence
difference between the two haemoglobin types is
greater than the species-specific differences. For
example, the Hb I chains of Anadara broughtoni
and A. trapezia agree at 82 % of positions, but
Hb I and Hb IIa of A. broughtoni are only 45 %
similar [108]. All these mussel haemoglobins are
rather unusually dissociation resistant: 1 m01l1
solutions of sodium iodide, propylurea or guanidine hydrochloride, which dissociate not only
human Hb A but also the more resistant fish haemoglobin, have no effect [17].
The subunits of the homodimer Hb I are
bound via contacts between the E and F helices;
the tetrameric Hb II is composed of two similarly
linked dimers. Whereas helices E and F point outwards in vertebrate haemoglobin dimers, they lie
internally in the haemoglobins of the Arcidae and
are responsible for the stability of the molecule
and for interaction between the subunits [108].
All these haemoglobins have relatively low O2
affinities, equally low Bohr effects and show no
further heterotropic interactions. They are
cooperative, with n values of 1.5 for the dimers
and 2.1 for the tetrameric forms. The responsible
homotropic interactions, however, are fundamentally different from those of vertebrate haemoglobins. In the latter, cooperativity and heterotropic modulation involve interaction between
heterodimers; in the Arcidae, the dimers themselves are already cooperative. Furthermore, vertebrate haemoglobins, with values of n :::; 3.0,
have more effective interaction than mussel haemoglobins [17, 37, 141].
The erythrocytes of other Arcidae, e.g. Noetia
ponderosa or Barbatia virescens, possess only
heterodimeric haemoglobins with subunits of
16 kDa. In addition to such heterodimers, the
related species Barbatia reeveana and B. lima also
contain polymeric haemoglobins of 430 kDa,
consisting of 12-14 subunits of 32 kDa with two
haem-bearing domains. Both domains apparently
arose by gene duplication and consequently show
high sequence similarity with each other and also