276
7 Respiratory Pigments
The O2 aff"mity of the haemocyanins is determined to a large extent by the pH, ion concentration and temperature. Because cooperativity is
influenced by the same factors, the Hill constants
for arthropod and mollusc haemocyanins usually
have values of only 3-4; however, extremely high
values may be achieved under optimal conditions, e.g. n = 7 for f3c-Hc of the edible snail
Helix pomatia, and n = 7-9 for the haemocyanin
of the spider Eurypelma californicum [108]. CO
also binds to the copper atoms of haemocyanin,
but in the arthropods with a much lower affinity
and cooperativity than does O2 [40]. An attempt
has been made many times to describe the cooperativity of the haemocyanins in terms of the
MWC model, but there are in fact few observations of relevant conformational changes. For
example, the haemocyanins of Helix pomatia and
Eurypelma californicum undergo changes in CuCu spacing and the number of Cu ligands on oxygenation which involve conformational changes
in the protein bridges between the copper atoms
[99, 108]. In terms of the molecular mass of a
respiratory pigment which is required for the
binding of one oxygen, the haemocyanins
(75 kDa for the arthropods and 55 kDa in molluscs) are significantly inferior to the haemoglobins (16 kDa). However, the arthropod haemocyanins in particular are modulated by allosteric
factors to an extent found in vertebrate, but
rarely in invertebrate, haemoglobins [115].
The O2 affinity of the haemocyanins is influenced by heterotropic interaction with H+, Cl-,
Ca 2 +, Mi+ and Na+. The transport of O 2 in whole
animals is in fact regulated mainly by pH changes
which affect haemocyanin directly and without
the damping effect of an intervening erythrocyte
membrane. Organic factors, such as lactate and
uric acid, also function as modulators in the crustaceans [108]. The pH effects on affinity and
cooperativity are independent, suggesting the
involvement of different protonated groups. The
pH dependence of the p50 of haemocyanins in
Limulus and several snails is given by a maximum
curve, e.g. in Buccinum undatum a maximal p50
value of 7.7 kPa is reached at pH 8.1; at pH
values below the maximum, an increase in pH
reduces the O2 affinity (a reverse Bohr effect).
This pH effect is so great in B. undatum that the
pigment cannot be completely saturated (reverse
Root effect). The biological sense of these
inverted pH effects is that under low oxygen
conditions acidic metabolic products can increase
the O2 affinity of haemocyanin [23]. Most haemocyanins have a normal Bohr effect which varies
greatly with the species and conditions. Particularly pronounced Bohr effects are found in some
arthropods with cp values of up to -1.3; normal
Root effects have been reported for Octopus dofleini and Panulirus interruptus [120]. Because the
oxygenation of haemocyanins is also an exothermic process, the O2 affinity is generally
reduced with an increase in temperature. The AH
values for various arthropod and mollusc haemocyanins were -13 to -47 kJ/mol at temperatures
between 15 and 25 °C [29]. It has been demonstrated by the detection of intracellular haemocyanins or the corresponding mRNA that the biosynthesis of haemocyanins occurs on membranebound ribosomes of the branchial glands of
cephalopods and on free ribosomes in so-called
cyanocytes of the arthropods. The latter cells are
found especially in tissues behind the eye of
Limulus polyphemus, in the hepato-pancreas and
close to the eye artery of the crustaceans Carcinus, Cancer and Astacus, and on the inner wall of
the heart of the spider Eurypelma [48]. Sequencing of haemocyanin mRNAs via the corresponding cDNAs is currently in progress [177]. Tracer
experiments indicate haemocyanin biosynthesis
in the mantle tissues of the snail Lymnaea stagnalis. This creature secretes a mixture of air and
haemolymph from the haemal pore to scare predators, losing in the process 1-3 mg haemocyanino Although the rate of synthesis subsequently
increases fivefold, it requires 14 days to make up
the loss. Iodinated species-specific haemocyanin
is degraded in the lobster Homarus americanus
with a half-life of 26 days.
7.3.1 Haemocyanins of the Arthropods
The arthropod haemocyanins are hexamers, or
multiples thereof, with subunits of about 75 kDa
bearing a single Oz-binding site. Various aggregates with differing sedimentation coefficients can
be distinguished: 16S (6-mer), 24S (12-mer), 36S
(24-mer) and 60S (48-mer). One or two of these
aggregation states predominates in each arthropod species, depending upon the number of different subunits and the possibilities for association. In the crabs, the 24S 12-mer is characteristic for the Astacura, Brachyura and Anomura; a
few species of this group have predominantly or
exclusively 6-mers. For example, in the semiterrestrial crab Ocypode quadrata, 56 % of the
haemocyanin is 6-mer and 44 % is 12-mer [77],
whereas the crab Uca urvillei and the crayfish
Jasus sp. have only 6-mers. Larger aggregates in
7 Respiratory Pigments
The O2 aff"mity of the haemocyanins is determined to a large extent by the pH, ion concentration and temperature. Because cooperativity is
influenced by the same factors, the Hill constants
for arthropod and mollusc haemocyanins usually
have values of only 3-4; however, extremely high
values may be achieved under optimal conditions, e.g. n = 7 for f3c-Hc of the edible snail
Helix pomatia, and n = 7-9 for the haemocyanin
of the spider Eurypelma californicum [108]. CO
also binds to the copper atoms of haemocyanin,
but in the arthropods with a much lower affinity
and cooperativity than does O2 [40]. An attempt
has been made many times to describe the cooperativity of the haemocyanins in terms of the
MWC model, but there are in fact few observations of relevant conformational changes. For
example, the haemocyanins of Helix pomatia and
Eurypelma californicum undergo changes in CuCu spacing and the number of Cu ligands on oxygenation which involve conformational changes
in the protein bridges between the copper atoms
[99, 108]. In terms of the molecular mass of a
respiratory pigment which is required for the
binding of one oxygen, the haemocyanins
(75 kDa for the arthropods and 55 kDa in molluscs) are significantly inferior to the haemoglobins (16 kDa). However, the arthropod haemocyanins in particular are modulated by allosteric
factors to an extent found in vertebrate, but
rarely in invertebrate, haemoglobins [115].
The O2 affinity of the haemocyanins is influenced by heterotropic interaction with H+, Cl-,
Ca 2 +, Mi+ and Na+. The transport of O 2 in whole
animals is in fact regulated mainly by pH changes
which affect haemocyanin directly and without
the damping effect of an intervening erythrocyte
membrane. Organic factors, such as lactate and
uric acid, also function as modulators in the crustaceans [108]. The pH effects on affinity and
cooperativity are independent, suggesting the
involvement of different protonated groups. The
pH dependence of the p50 of haemocyanins in
Limulus and several snails is given by a maximum
curve, e.g. in Buccinum undatum a maximal p50
value of 7.7 kPa is reached at pH 8.1; at pH
values below the maximum, an increase in pH
reduces the O2 affinity (a reverse Bohr effect).
This pH effect is so great in B. undatum that the
pigment cannot be completely saturated (reverse
Root effect). The biological sense of these
inverted pH effects is that under low oxygen
conditions acidic metabolic products can increase
the O2 affinity of haemocyanin [23]. Most haemocyanins have a normal Bohr effect which varies
greatly with the species and conditions. Particularly pronounced Bohr effects are found in some
arthropods with cp values of up to -1.3; normal
Root effects have been reported for Octopus dofleini and Panulirus interruptus [120]. Because the
oxygenation of haemocyanins is also an exothermic process, the O2 affinity is generally
reduced with an increase in temperature. The AH
values for various arthropod and mollusc haemocyanins were -13 to -47 kJ/mol at temperatures
between 15 and 25 °C [29]. It has been demonstrated by the detection of intracellular haemocyanins or the corresponding mRNA that the biosynthesis of haemocyanins occurs on membranebound ribosomes of the branchial glands of
cephalopods and on free ribosomes in so-called
cyanocytes of the arthropods. The latter cells are
found especially in tissues behind the eye of
Limulus polyphemus, in the hepato-pancreas and
close to the eye artery of the crustaceans Carcinus, Cancer and Astacus, and on the inner wall of
the heart of the spider Eurypelma [48]. Sequencing of haemocyanin mRNAs via the corresponding cDNAs is currently in progress [177]. Tracer
experiments indicate haemocyanin biosynthesis
in the mantle tissues of the snail Lymnaea stagnalis. This creature secretes a mixture of air and
haemolymph from the haemal pore to scare predators, losing in the process 1-3 mg haemocyanino Although the rate of synthesis subsequently
increases fivefold, it requires 14 days to make up
the loss. Iodinated species-specific haemocyanin
is degraded in the lobster Homarus americanus
with a half-life of 26 days.
7.3.1 Haemocyanins of the Arthropods
The arthropod haemocyanins are hexamers, or
multiples thereof, with subunits of about 75 kDa
bearing a single Oz-binding site. Various aggregates with differing sedimentation coefficients can
be distinguished: 16S (6-mer), 24S (12-mer), 36S
(24-mer) and 60S (48-mer). One or two of these
aggregation states predominates in each arthropod species, depending upon the number of different subunits and the possibilities for association. In the crabs, the 24S 12-mer is characteristic for the Astacura, Brachyura and Anomura; a
few species of this group have predominantly or
exclusively 6-mers. For example, in the semiterrestrial crab Ocypode quadrata, 56 % of the
haemocyanin is 6-mer and 44 % is 12-mer [77],
whereas the crab Uca urvillei and the crayfish
Jasus sp. have only 6-mers. Larger aggregates in