are related to tetrameric aggregation [139]. The
haemoglobin of the semi-aquatic snake Liophis
miliaris dissociates to dimers on oxygenation.
Sequence comparisons with human Hb A show
five amino acid differences in the a l j32 contact
region, including the exchanges j3G3Glu ~ Val
and j3CD2Glu ~ Thr. It is probably this loss of
two negative charges that is responsible for the
lower stability of binding between the dimers
[45, 118]. In fish, there are haemoglobins whose
properties are not in total agreement with the
MWC mode. They show Hill constant values as
low as 1.0, particularly in acid conditions, and this
indicates a marked inhibition of the T ~ R transition [21]. Hill constants under 1.0 are also possible if the O2 affinities of th a- and j3-chains differ
greatly [139]. According to the MWC model,
heterotropic ligands should change only the L
values; KT and KR should remain constant. However, in the case of the haemoglobins of several
fish species and mammals, including man, organophosphates not only increase the L value but
also reduce KT; KR remains unchanged and the
T ~ R transition is hindered. This effect can be
explained by a three-state model, according to
which there exists, in addition to T and R, a third
state, S, in which the low affinity state T is stabilized by the organophosphate [75]. More recent
cooperativity models are based upon the MWC
model but take into account the thermodynamic
equivalence of the salt bridges between the
dimers, as postulated by Perutz, and allow a
quantitative description of homotropic and heterotropic interactions in vertebrate haemoglobin
[98].
The only vertebrate haemoglobins which are
not built on the tetramer model described above
are from the agnathans, the lampreys (Petromyzontoidae) and the hagfish (Myxinoidae). The
marine lamprey Petromyzon marinus has a principal haemoglobin (Hb II) and two electrophoretically distinct subsidiary components (Hb I and
Hb III), all of which are monomeric in the oxygenated state but can aggregate to dimers after
oxygenation at weakly acidic pH:
G2 + 202 ~ 2G02.
This change in the aggregation state with oxygenation or deoxygenation may be considered an
extreme case of conformational change as the
result of homotropic interaction; that is why these
haemoglobins also show cooperativity. The reason why they cannot form tetramers can be found
in their amino acid sequence. All three haemoglobins lack nine amino acids in the GH region,
7.1.3 HeterotropicInteractions
255
and in several positions important for a l j3l contacts there are more strongly polar amino acids to
be found than in the other vertebrates; on the
other hand, the a l j32 contacts are more similar to
those of the mammals. Thus, these haemoglobin
chains can be linked by contacts which correspond to the a l j32 contacts of the tetrameric vertebrate haemoglobins, but the a l j3l contacts necessary for tetramer formation cannot be produced. The haemoglobins of the marine lamprey
(P. marin us) and the river lamprey (Lampetraf/uviatilis) differ at only three to five positions. In
contrast, the haemoglobin sequences of the common hagfish Myxine glutinosa and the Petromyzontidae differ by 87-90 amino acids. The haemoglobin of Eptatretus stoutii (Myxinoidae)
appears to exist as monomers in the presence or
absence of oxygen [65, 67]; on the other hand,
the deoxyhaemoglobins of the related species
E. burgeri and E. cirrhatus have a tendency to
aggregate at low pH and high protein concentration [20].
The a l j32 contacts in the haemoglobins of the
Petromyzontidae may be looked upon as the first
evolutionary step from the monomeric globins,
resembling myoglobin, to the tetrameric vertebrate haemoglobins. This first step already allowed
cooperative and allosteric interactions. In the following evolutionary step, two chains arose after a
gene duplication; these could form heterodimers
and- tetramers, and allowed finer grading of the
homo tropic and heterotropic interactions. The
"discovery" and refinement of cooperativity and
the heterotropic modulation of vertebrate haemoglobins should be considered in the light of the
increase in metabolism, which required high and
constant oxygen partial pressures in the tissues as
independent as possible from O2 use and CO2
production. Further gene duplications resulted in
the appearance of many a- and j3-like chains,
from which a whole spectrum of haemoglobins
with varying properties could be produced. Finer
adaptations to various internal and external
conditions consequently became possible. The
course of haemoglobin evolution can be illustrated by a molecular genealogical tree (Fig. 7.6).
7.1.3 Heterotropic Interactions
Vertebrate haemoglobins show an extraordinary
variety of functional characteristics, such as their
intrinsic O2 affinity and their sensitivity to allosteric modulation, which allow adaption to very
different internal and external conditions. Allos-
haemoglobin of the semi-aquatic snake Liophis
miliaris dissociates to dimers on oxygenation.
Sequence comparisons with human Hb A show
five amino acid differences in the a l j32 contact
region, including the exchanges j3G3Glu ~ Val
and j3CD2Glu ~ Thr. It is probably this loss of
two negative charges that is responsible for the
lower stability of binding between the dimers
[45, 118]. In fish, there are haemoglobins whose
properties are not in total agreement with the
MWC mode. They show Hill constant values as
low as 1.0, particularly in acid conditions, and this
indicates a marked inhibition of the T ~ R transition [21]. Hill constants under 1.0 are also possible if the O2 affinities of th a- and j3-chains differ
greatly [139]. According to the MWC model,
heterotropic ligands should change only the L
values; KT and KR should remain constant. However, in the case of the haemoglobins of several
fish species and mammals, including man, organophosphates not only increase the L value but
also reduce KT; KR remains unchanged and the
T ~ R transition is hindered. This effect can be
explained by a three-state model, according to
which there exists, in addition to T and R, a third
state, S, in which the low affinity state T is stabilized by the organophosphate [75]. More recent
cooperativity models are based upon the MWC
model but take into account the thermodynamic
equivalence of the salt bridges between the
dimers, as postulated by Perutz, and allow a
quantitative description of homotropic and heterotropic interactions in vertebrate haemoglobin
[98].
The only vertebrate haemoglobins which are
not built on the tetramer model described above
are from the agnathans, the lampreys (Petromyzontoidae) and the hagfish (Myxinoidae). The
marine lamprey Petromyzon marinus has a principal haemoglobin (Hb II) and two electrophoretically distinct subsidiary components (Hb I and
Hb III), all of which are monomeric in the oxygenated state but can aggregate to dimers after
oxygenation at weakly acidic pH:
G2 + 202 ~ 2G02.
This change in the aggregation state with oxygenation or deoxygenation may be considered an
extreme case of conformational change as the
result of homotropic interaction; that is why these
haemoglobins also show cooperativity. The reason why they cannot form tetramers can be found
in their amino acid sequence. All three haemoglobins lack nine amino acids in the GH region,
7.1.3 HeterotropicInteractions
255
and in several positions important for a l j3l contacts there are more strongly polar amino acids to
be found than in the other vertebrates; on the
other hand, the a l j32 contacts are more similar to
those of the mammals. Thus, these haemoglobin
chains can be linked by contacts which correspond to the a l j32 contacts of the tetrameric vertebrate haemoglobins, but the a l j3l contacts necessary for tetramer formation cannot be produced. The haemoglobins of the marine lamprey
(P. marin us) and the river lamprey (Lampetraf/uviatilis) differ at only three to five positions. In
contrast, the haemoglobin sequences of the common hagfish Myxine glutinosa and the Petromyzontidae differ by 87-90 amino acids. The haemoglobin of Eptatretus stoutii (Myxinoidae)
appears to exist as monomers in the presence or
absence of oxygen [65, 67]; on the other hand,
the deoxyhaemoglobins of the related species
E. burgeri and E. cirrhatus have a tendency to
aggregate at low pH and high protein concentration [20].
The a l j32 contacts in the haemoglobins of the
Petromyzontidae may be looked upon as the first
evolutionary step from the monomeric globins,
resembling myoglobin, to the tetrameric vertebrate haemoglobins. This first step already allowed
cooperative and allosteric interactions. In the following evolutionary step, two chains arose after a
gene duplication; these could form heterodimers
and- tetramers, and allowed finer grading of the
homo tropic and heterotropic interactions. The
"discovery" and refinement of cooperativity and
the heterotropic modulation of vertebrate haemoglobins should be considered in the light of the
increase in metabolism, which required high and
constant oxygen partial pressures in the tissues as
independent as possible from O2 use and CO2
production. Further gene duplications resulted in
the appearance of many a- and j3-like chains,
from which a whole spectrum of haemoglobins
with varying properties could be produced. Finer
adaptations to various internal and external
conditions consequently became possible. The
course of haemoglobin evolution can be illustrated by a molecular genealogical tree (Fig. 7.6).
7.1.3 Heterotropic Interactions
Vertebrate haemoglobins show an extraordinary
variety of functional characteristics, such as their
intrinsic O2 affinity and their sensitivity to allosteric modulation, which allow adaption to very
different internal and external conditions. Allos-