COMMUNICATION
ATP-induced Tetramerization and Cooperativity in Hemoglobin of
Lower Vertebrates*
Carlos F. S.
Bonafe,
Adriana Y.
Matsukuma, and
Maria
S. A.
Matsuura
From the Departamento de Bioquímica, Instituto de Biologia,
Universidade Estadual de Campinas, 13083-970, Campinas, São
Paulo, Brazil
 |
ABSTRACT |
The importance of intraerythrocytic organic
phosphates in the allosteric control of oxygen binding to vertebrate
hemoglobin (Hb) is well recognized and is correlated with
conformational changes of the tetramer. ATP is a major allosteric
effector of snake Hb, since the absence of this nucleotide abolishes
the Hb cooperativity. This effect may be related to the molecular
weight of about 32,000 for this Hb, which is compatible with the
dimeric form. ATP induces a pH-dependent tetramerization of
deoxyHb that leads to the recovery of cooperativity. This
phenomenon may be partially explained by two amino acid replacements in
the
chains (CD2 Glu-43
Thr and G3 Glu-101
Val), which
result in the loss of two negative charges at the
1
2 interface and favors the
dissociation into dimers. The ATP-dependent dimer
tetramer may be physiologically important among ancient animal groups
that have similar mutations and display variations in blood pH that are
governed by these animals' metabolic state. The enormous loss of free
energy of association that accompanies Hb oxygenation, and which is
also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the
transition from a myoglobin-like protein to an allosteric one may be of
evolutionary significance.
 |
INTRODUCTION |
In vertebrates, hemoglobin (Hb) exists as a tetramer in its
intraerythrocytic environment, and it is this form that is involved in
the classic structural change from a low to a high O2
affinity molecule in the presence of increasing O2
concentrations. This phenomenon, known as cooperativity, is reflected
in the sigmoidal shape of the O2 saturation curve.
Protons and organic phosphate are important in the physiological
transport of O2 in most vertebrate groups, since they
stabilize the low affinity form of Hb (1, 2).
Previous studies have demonstrated an oxygen-induced dissociation of
snake Hb at physiological pH and Hb concentration, as well as in the
presence of high levels of organic phosphate (3). The structural basis
of this phenomenon is the replacement of amino acid residues
-CD2-43 and
-G3-101 at the
1
2
interface which is responsible for tetramer stabilization. These key
residues, normally both glutamic acid, are replaced by threonine and
valine, respectively, in snake Hb (4). This loss of negative charges would disturb the interface contact, leading to a pronounced tendency of Hb to dissociate into dimers. Since these residues are also replaced
in most hemoglobins from ectothermic animals (5-9), this suggests that
a dissociation of Hb occurs during oxygenation. The physiological role
of such Hb dissociation is considered in the present investigation.
 |
EXPERIMENTAL PROCEDURES |
Hemoglobin Preparation--
Adult snakes of both sexes weighing
200-400 g were obtained from the Instituto Butantã (São
Paulo) and were kept in the laboratory until bleeding. The hemolysate
was prepared as described by Rossi-Fanelli and Antonini (10) and was
freed of salts and small organic molecules by passage through a
Sephadex G-25 column (2.0 × 90 cm) equilibrated with 1 mM Tris-HCl, pH 9.0 (11), to produce "stripped" Hb.
Measurement of Redox Potentials--
The redox titrations were
carried out according to Antonini et al. (12). Five
milliliters of Hb solution (140 µM as heme) were
deoxygenated in a tonometer and then transferred anaerobically, with
continuous flush of N2, to the titration half-cell, which contained 0.1 M Tris-HCl plus 0.1 M NaCl (pH
range: 7.0-8.0). Thionin was added as a mediator in a molar ratio to
protein of 2-4%. The oxidation of deoxyHb was performed by the
stepwise addition of a degassed solution of 5 mM potassium
ferricyanide. The measured electrode potentials were refereed to the
normal hydrogen electrode (13). The oxidation-reduction potential at
50% of oxidation provided the midpoint potential
(E1/2).
O2-Hb Equilibrium--
The experiments were
performed at 20 °C in 0.1 M Tris-HCl buffer of different
pH values containing 0.1 M NaCl, using a
tonometric-spectrophotometric method (14). The protein concentration
was 80 µM (as heme).
 |
RESULTS AND DISCUSSION |
To gain insight into the possible physiological role of pH and ATP
in the subunit assembly of snake (Helicops modestus) Hb, we
investigated the Hb-O2 equilibrium as a function of proton concentration in the presence or absence of ATP (Fig.
1). Stripped snake Hb showed a high
affinity for O2 and no allosterism, in accordance with a
molecular mass compatible with the dimeric form (3, 10, 15). In the
presence of organic phosphates, the molecule became cooperative
(nH = 2) with a low O2 affinity at a pH up to
7.4. With increasing pH, the Hb gradually lost cooperativity, suggesting a weakening of the electrostatic interaction between ATP and
Hb. As a result, the latter tended to assume the properties of stripped
Hb. The pH sensitivity cannot be attributed exclusively to a classic
Bohr effect in view of the dimerization process that is also
present.

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Fig. 1.
Effect of ATP on the O2
equilibrium of H. modestus Hb at different pH values.
The buffer used was 0.1 M Tris-HCl containing 0.1 M NaCl, at 20 °C, and the Hb concentration was 80 µM as heme. , stripped Hb; , Hb in the presence of
1.0 mM ATP. Inset, nH
values derived from a Hill plot.
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|
Based on these unusual findings, we investigated the redox potential of
snake Hb under the same conditions as those used for the O2
equilibrium curves in order to better understand the Hb properties in
the presence of ATP at different pH values. This approach, applied to
either tetrameric Hbs or myoglobins, has been employed to show the
conversion of the deoxy to the met form and its close correlation with
oxygenation equilibrium curves, since the potentiometric curves share
similarities with the equilibrium ligand binding curves for Hb (12,
16-20). Fig. 2A illustrates that snake Hb had a peculiar behavior in this experiment. The redox
potential of stripped Hb did not change with a pH of up to 7.6, but
decreased at higher pH values. The resulting curve was similar to that
of myoglobin and corroborated our expectation that stripped Hb is
dissociated even in the deoxygenated form. The progressive decrease in
Eh observed with increasing pH in both stripped
and ATP-Hb is correlated to the extend of water ionization on the sixth
coordinate of heme iron (18). ATP dramatically changed the redox
equilibrium profile. In the presence of ATP, the
Eh value at pH up to 7.22 was constant and
higher than in the absence of the nucleotide. However, the
Eh value decreased sharply in the pH range of
7.22-7.38. This observation is consistent for a tetrameric Hb in which
the classic allosteric model is found. The redox equilibrium curve at
pH > 7.80 superposed the stripped Hb curve, indicating the
complete release of ATP from its binding site. In the pH range of
7.38-7.80, the redox potential presented a curve compatible with
equilibrium between dimers and ATP-bound tetramers. From Fig.
2A we estimated the quantitative contribution of the
different molecular forms of Hb (Fig. 2B). The dissociation of tetrameric Hb into dimers was observed primarily between pH 7.38 and
7.55 and varied from 0 to 80%.

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Fig. 2.
A, effect of ATP on the
oxidation-reduction equilibrium of H. modestus Hb at
different pH values. , stripped Hb; , Hb in the presence of 1.0 mM ATP. The buffer used was 0.1 M Tris-HCl
containing 0.1 M NaCl at 20 °C, and the Hb concentration
was 140 µM as heme. The dashed line indicates
the theoretical tetramer behavior. A, inset: Hill
plots of the oxidation-reduction equilibrium curves for H. modestus stripped Hb (open symbols) and in the presence
of 1.0 mM ATP (closed symbols). B,
estimation of the proportion of dimeric and tetrameric Hb in the
presence of ATP as a function of pH, based on the above figure. For
each pH value, the fraction corresponding to the tetramer,
F 2 2, and dimer,
F , was calculated based on the theoretical data for
the tetramer (dashed line of Eh curve
in the presence of ATP, which corresponds to the theoretical behavior
if ATP were bound) and on experimental data for the dimer
(Eh curve in the absence of ATP):
F 2 2 = (Eh Eh)/(Eh Eh 2 2);
F = 1 F 2 2.
|
|
The inset in Fig. 2A shows the corresponding Hill
plots of the redox equilibrium. At pH 7.0, the oxidated Hb retained its tetrameric form, indicating that ATP remains bound independently of the
degree of oxidation. At pH 7.80, the Hb dissociated and had the same
nH values as stripped Hb. However, at pH 7.38, the biphasic behavior indicated that above 50% of oxidation, R-met Hb
became very unstable and immediately dissociated into dimers.
The substantial differences in the Hb properties described above assume
a great significance when the physiological state of ectothermic
vertebrates are considered. Several studies have reported large blood
pH changes when ectothermic animals are subjected to different
temperature or stress conditions (21-23). In such situations, the
proton concentration would be particularly important in influencing the
binding of ATP to Hb, thereby altering the protein's O2
affinity. These functional properties may be present in a large array
of animals from related groups, since the replacement of amino acid
residues at key positions of
1
2 contact
is present (5-9).
Fig. 3 proposes a general model for
O2 transport by snake and other related vertebrate Hbs in
which the ATP plays a central role. In the dormancy state, when low
O2 transport is required and the blood pH is increased, the
Hb exists in a dimeric form that acts as a reserve supply of
O2 in a manner similar to myoglobin. In stress or high
activity, the decrease in pH promotes ATP-induced tetramerization and
allosterism, thereby resulting in a significant O2 release.
Thus in this dynamic interchange, ATP and pH changes serve to integrate
the physiology of O2 supply. This novel model provides new
insight into O2 transport when compared with higher vertebrates in which the cooperative ligand binding of Hb is based on a
switching between quaternary states of the Hb tetramer with different
O2 affinities (24).
From a thermodynamic aspect, the classic T-R model of
Monod-Changeux-Wyman (MCW model) does not take into account the free energy of association between 
dimers (25). This situation was
considered by Weber (26), who demonstrated that O2 binding to human Hb is a first order reaction that is inconsistent with the
two-state model. It is noteworthy that the dissociation of snake Hb is
an extreme example of decreasing the Gibbs energy of association
between dimers, since progressive oxygenation is linked with
dissociation into more reactive dimeric species. Thus, the presence of
the classic R state is theoretical and difficult to detect
experimentally (Fig. 1), except in metHb obtained by redox potential
experiments (Fig. 2A). In Fig.
4, we propose a diagram of the Gibbs free
energy of O2 binding with snake ATP-Hb in comparison with
stripped human Hb. The most striking feature is the inversion of the
free energy of association between oxygenated dimers despite the
presence of ATP. Moreover, the binding of the first/second
O2 molecule results in a much higher affinity of tetrameric
snake Hb to further O2 binding than is the case with human
Hb.

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Fig. 4.
Gibbs free energy levels of Hb subunit
association and oxygen binding by human (A) and snake
(B) Hb. D = dimer; T = tetramer; X = O2.
G(n) = free energy of subunit association with
"n" molecules of O2.
G(2,n) and G(4,n) = free energy of O2 binding of dimer and tetramer,
respectively, with "n 1" molecules of
O2.
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|
The evolutionary adaptation, study of Hb structure and function, has
been extensively discussed (27, 28). The Agnatha, lampreys, and
hagfish, the most primitive group of vertebrates, have monomeric Hb in
which the O2 transport mechanism is accomplished by a dimer
monomer transition. This disaggregation leads to a higher affinity
state and allows cooperative behavior (29-31). The singular properties
of snake Hb reported here may point to the origin of a stable dimeric
molecule that could have important evolutionary implications for
heme-heme interactions. The 
dimeric form may represent an
intermediary evolutionary stage of the classic allosterism of
vertebrate Hbs, where ATP serves as a central allosteric mediator. The
molecular properties of such Hbs may reflect the physiological
functions of ectothermic Hb, particularly the adaptations to exogenous
and endogenous factors such as ambient hypoxia, temperature, activity,
and dormancy. Finally, the mechanism of dimer-tetramer transitions
during O2 transport may represent an intermediate stage of
evolution to the stable tetrameric Hb found in higher vertebrates.
 |
ACKNOWLEDGEMENT |
We are grateful to Prof. Stephen Hyslop for
helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP), Proc.
95-1245/7; Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq); and Fundação de Amparo ao Ensino
e à Pesquisa (FAEP-UNICAMP) e Serviço de Apoio ao Estudante
(SAE-UNICAMP).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. de
Bioquímica, Instituto de Biologia, Universidade Estadual de
Campinas, 13083-970, Campinas, SP, Brazil. Tel.: 55-19-788-7953; Fax:
55-19-289-3124; E-mail: bonafe{at}obelix.unicamp.br.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.