268
7 Respiratory Pigments
The extracellular baemoglobins of the annelids
are giant molecules whose structure can be analysed with the electron microscope. They have the
form of two superimposed hexagons with moreor-less spherical subunits. For technical reasons,
previous studies reported widely varying values
for the dimensions of these molecules. More
recent investigations consistently give a diameter
of 30 nm and a height of 20 nm. The molecular
mass is 3.6-3.8 MDa [56, 79]. In addition to
molecules with the described dimensions, the
polychaetes Ophelia bicornis and Euzonus mucronata of the family Ophelidae contain aggregates with a two- to threefold larger mass [108].
In mildly alkaline conditions, the molecules
dissociate into fragments of varying size but
always include a fragment that is 1I12th of the original mass. Whereas Ca2+ stabilizes the molecular structure, ethylene diaminetetraacetic acid
(EDT A) promotes dissociation. A few species
show reversible dissociation (Dina dubia, Haemopis sanguisuga), and in Eunice aphroditis an
association-dissociation equilibrium exists over a
wide pH range around the neutral point. Dissociation of the native haemoglobin molecule can
also occur at acidic or neutral pH values upon
addition of urea, perchlorate or certain other anions[108]. For a long time there was controversy
over just how many different polypeptides exist in
extracellular haemoglobins and chlorocruorins of
the annelids, and which are the stoichiometric
relationships between these types of polypeptides. It is now agreed that the haemoglobins of the
polychaetes and oligochaetes consist of four types
of haem-bearing 16-kDa chains, three of which
are linked by disulphide bridges. There are, in
addition, at least two types of non-haem-bearing
30-kDa chains known as "linkers". The situation
in the leech Macrobdella decora is somewhat different. SDS-gel chromatography under nonreducing conditions in this case gives three types
of subunit: monomers of about 17 kDa, nonreducible dimers of 30 kDa, and reducible haembearing dimers of 34 kDa. According to the Nterminal sequences, at least five different polypeptides are present [79]. Amino acid sequences
have been obtained for all four haem-bearing
chains of the polychaete Tylorrhynchus heterochaetus and of the common earthworm Lumbricus terrestris, and for the monomeric subunits of
the freshwater oligochaete Tubifex tubifex and of
the earthworm Pheretima sieboldi. The ten haembearing chains agree in 30-50 % of positions; of
the 25 invariant amino acids, 9 are conserved in
the human B-chain [56, 154]. Linkers 1 and 2 of
Tylorrhyncus and one linker of the deep-sea pogonophoran Lamellibrachia have also been sequenced; they show agreement in 23-27 % of
their 224-253 positions but show no relationship
to the haemoglobin sequences [160]. The only
annelid haemoglobin gene so far to have been
sequenced, the gene for chain III (or c) of Lumbricus terrestris, contains two introns of 1344 and
1169 bp in positions homologous to those in vertebrate haemoglobin genes [76].
Because of the existence of polypeptides without haem, the haem (2.6 ± 0.4 %) and iron
(0.23 ± 0.01 %) contents of the annelid haemoglobins are both very low compared with those of
other haemoglobins; this results in the unusually
high mean molecular mass of 23-26 kDa per
haem [175]. The nomenclature of the haembearing chains is inconsistent: in Tylorrhynchus,
the monomeric chains are labelled as I and the
subunits of the trimers as IIA-IIC; the corresponding designations in Lumbricus are I or d
and II-IV or a-c. Because of the greater sequence
similarity pairwise, the designations a, A, b, B
were suggested more recently for the four chains
[56]. Accordingly, the monomeric subunit is termed a (Tylorrhynchus I, Lumbricus I or d), and
the subunits of the trimers are called A (Tylorrhynchus IIA, Lumbricus II or b), b (Tylorrhynchus IIC, Lumbricus III or c) and B (Tylorrhynchus lIB, Lumbricus IV or a). Despite many
investigations with the most advanced electron
microscopic techniques, the quarternary structure
of the annelid haemoglobins has not yet been
unambiguously defined. One model that is
acceptable to many workers in the field is similar
to the "bracelet" model proposed by Vinogradov
in 1986; in this, 12 complexes with the composition (aAbB)4 are bound together via 24 haem-free
subunits to form two sets of six linked rings. The
value of 192 haem groups per molecule which
emerges from this model is somewhat larger than
that calculated from the haem content and molecular mass [56, 174].
The extracellular annelid haemoglobins show
cooperativity. For earthworm haemoglobin, Hill
constants, n, have been recorded as between 2.5
and 7.9, depending upon the pH; Ca 2 + shifts the
pH for nmax from 8.1 to 7.6, and at the same time
increases both the O2 affinity and the Bohr effect.
The 1/12th fragments also show cooperativity;
although the n values for these fragments are
always lower than for native haemoglobin, the
homotropic interaction nevertheless appears to
lie mainly, or entirely, within this 1/12th structure
[50, 108, 176]. The O2 affinities of annelid
7 Respiratory Pigments
The extracellular baemoglobins of the annelids
are giant molecules whose structure can be analysed with the electron microscope. They have the
form of two superimposed hexagons with moreor-less spherical subunits. For technical reasons,
previous studies reported widely varying values
for the dimensions of these molecules. More
recent investigations consistently give a diameter
of 30 nm and a height of 20 nm. The molecular
mass is 3.6-3.8 MDa [56, 79]. In addition to
molecules with the described dimensions, the
polychaetes Ophelia bicornis and Euzonus mucronata of the family Ophelidae contain aggregates with a two- to threefold larger mass [108].
In mildly alkaline conditions, the molecules
dissociate into fragments of varying size but
always include a fragment that is 1I12th of the original mass. Whereas Ca2+ stabilizes the molecular structure, ethylene diaminetetraacetic acid
(EDT A) promotes dissociation. A few species
show reversible dissociation (Dina dubia, Haemopis sanguisuga), and in Eunice aphroditis an
association-dissociation equilibrium exists over a
wide pH range around the neutral point. Dissociation of the native haemoglobin molecule can
also occur at acidic or neutral pH values upon
addition of urea, perchlorate or certain other anions[108]. For a long time there was controversy
over just how many different polypeptides exist in
extracellular haemoglobins and chlorocruorins of
the annelids, and which are the stoichiometric
relationships between these types of polypeptides. It is now agreed that the haemoglobins of the
polychaetes and oligochaetes consist of four types
of haem-bearing 16-kDa chains, three of which
are linked by disulphide bridges. There are, in
addition, at least two types of non-haem-bearing
30-kDa chains known as "linkers". The situation
in the leech Macrobdella decora is somewhat different. SDS-gel chromatography under nonreducing conditions in this case gives three types
of subunit: monomers of about 17 kDa, nonreducible dimers of 30 kDa, and reducible haembearing dimers of 34 kDa. According to the Nterminal sequences, at least five different polypeptides are present [79]. Amino acid sequences
have been obtained for all four haem-bearing
chains of the polychaete Tylorrhynchus heterochaetus and of the common earthworm Lumbricus terrestris, and for the monomeric subunits of
the freshwater oligochaete Tubifex tubifex and of
the earthworm Pheretima sieboldi. The ten haembearing chains agree in 30-50 % of positions; of
the 25 invariant amino acids, 9 are conserved in
the human B-chain [56, 154]. Linkers 1 and 2 of
Tylorrhyncus and one linker of the deep-sea pogonophoran Lamellibrachia have also been sequenced; they show agreement in 23-27 % of
their 224-253 positions but show no relationship
to the haemoglobin sequences [160]. The only
annelid haemoglobin gene so far to have been
sequenced, the gene for chain III (or c) of Lumbricus terrestris, contains two introns of 1344 and
1169 bp in positions homologous to those in vertebrate haemoglobin genes [76].
Because of the existence of polypeptides without haem, the haem (2.6 ± 0.4 %) and iron
(0.23 ± 0.01 %) contents of the annelid haemoglobins are both very low compared with those of
other haemoglobins; this results in the unusually
high mean molecular mass of 23-26 kDa per
haem [175]. The nomenclature of the haembearing chains is inconsistent: in Tylorrhynchus,
the monomeric chains are labelled as I and the
subunits of the trimers as IIA-IIC; the corresponding designations in Lumbricus are I or d
and II-IV or a-c. Because of the greater sequence
similarity pairwise, the designations a, A, b, B
were suggested more recently for the four chains
[56]. Accordingly, the monomeric subunit is termed a (Tylorrhynchus I, Lumbricus I or d), and
the subunits of the trimers are called A (Tylorrhynchus IIA, Lumbricus II or b), b (Tylorrhynchus IIC, Lumbricus III or c) and B (Tylorrhynchus lIB, Lumbricus IV or a). Despite many
investigations with the most advanced electron
microscopic techniques, the quarternary structure
of the annelid haemoglobins has not yet been
unambiguously defined. One model that is
acceptable to many workers in the field is similar
to the "bracelet" model proposed by Vinogradov
in 1986; in this, 12 complexes with the composition (aAbB)4 are bound together via 24 haem-free
subunits to form two sets of six linked rings. The
value of 192 haem groups per molecule which
emerges from this model is somewhat larger than
that calculated from the haem content and molecular mass [56, 174].
The extracellular annelid haemoglobins show
cooperativity. For earthworm haemoglobin, Hill
constants, n, have been recorded as between 2.5
and 7.9, depending upon the pH; Ca 2 + shifts the
pH for nmax from 8.1 to 7.6, and at the same time
increases both the O2 affinity and the Bohr effect.
The 1/12th fragments also show cooperativity;
although the n values for these fragments are
always lower than for native haemoglobin, the
homotropic interaction nevertheless appears to
lie mainly, or entirely, within this 1/12th structure
[50, 108, 176]. The O2 affinities of annelid