Download Review Hemoglobin function under extreme life conditions

Eur. J. Biochem. 223, 309-317 (1994)
0 FEBS 1994
Review
Hemoglobin function under extreme life conditions
Maria E. CLEMENTI’, Saverio G. CONDO’, Massimo CASTAGNOLA’.’ and Bruno GIARDINA,
’
Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Italy
* Institute of Chemistry and Clinical Chemistry, Faculty of Medicine and CNR Center ‘Chimica dei recettori e delle molecole
biologicamente attive’ , Catholic University, Rome, Italy
(Received December 15, 1993March 22, 1994)
-
EJB 93 1858/0
Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. Hence, a
thermodynamic analysis of oxygen binding with hemoglobins from different species reveals a series
of adaptive mechanisms which are based on the thermodynamic connection between the binding of
heterotropic effectors and the reaction with oxygen.
The examples reported, from fishes to human fetus, illustrate how evolution can alter the structural basis of the heterotropic interactions to optimize the oxygenation-deoxygenation cycle in
dependence of the physiological needs of the particular organisms. Moreover they show that a
thermodynamic analysis of the reaction with oxygen overcomes the meaning of a detailed structural
and functional characterization going deeper into the physiology of the specific organism.
Respiratory pigments directly link external conditions
with body requirements and are therefore interesting systems
for studying the effect of the environment on molecular evolution. Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute
their primary function under extreme environmental conditions. Hence, the hemoglobins have experienced a major evolutionary pressure which has led to the development of a
number of complex regulatory mechanisms operative at the
molecular level and designed to fulfil the physiological
requirements of a given species.
All vertebrate hemoglobins exhibit a marked degree of
cooperativity between subunits or ‘heme- heme’ interaction
(homotropic interactions) which enables maximum oxygen
unloading at relatively high oxygen tension. In the simple
model, cooperativity in oxygen binding is achieved through
the conformational transition, between the deoxy-low-affinity state (or T state) and the oxy-high-affinity state (or R
state), which accounts for the sigmoidal shape of the oxygen
binding curve. In addition, the oxygen affinity of hemoglobin
is affected by several metabolic effectors (heterotropic interactions) such as chloride, protons (Bohr effect), CO, and
organic phosphates. Under physiological conditions, in fact,
all these effectors bind preferentially to the deoxy conformation (T state) of hemoglobin thereby shifting the allosteric
Correspondence to B . Giardina, Istituto di Chimica, Universith
Cattolica del “Sacro Cuore”, Largo F. Vito 1, 1-00168 Roma, Italy
Abbreviations. GriP,, 2,3-bis-phosphoglycerate;InsP,, inositol
hexakisphosphate ; InsP,, inositol pentaphosphate ; P,,, partial pressure of oxygen required to give 50% of the heme molecules saturated with oxygen; A H , overall heat of oxygenation calculated from
the van’t Hoff equation; HbA, human adult hemoglobin; HbF, human fetal hemoglobin.
equilibrium ( T o R ) towards the T state and lowering the
overall 0, affinity of the molecule.
The modulation of function induced by these effectors
has important physiological effects. For example, at the level
of respiring tissues, the decrease of 0, affinity brought about
by the increase in proton activity (alkaline Bohr effect) allows a more efficient unloading of 0, and contributes to the
neutralization of protons produced by CO, and lactic acid.
Another important feature of the reaction of hemoproteins with 0, is its temperature dependence which is governed by the associated overall enthalpy change (AH). Oxygen binding to mammalian hemoglobins is generally exothermic ( A H negative) so that a decrease in temperature induces
an increase of the 0, affinity.
In the case of a simple hemoprotein such as myoglobin
the heat released on binding 0, is generally in the region
of -62.8 kJ mol-I, but for HbA this release of heat is reduced to about -33.5 kJ mol-’ because of the compensating
effect of other 0,-linked processes. In this respect it is informative to correlate the different contributions to the thermal
effects measured when 0, binds to hemoglobin. These may
be summarized as: (a) intrinsic heat of oxygenation, namely
the heat involved in the binding of 0, to the heme iron; (b)
heat of ionization of 0,-linked ionizable groups (Bohr
groups) which is always endothermic ( A H positive); (c) heat
of 0, solubilization (-12.5 kJ mol-’, exothermic); (d) heat
associated with the T+R allosteric transition and (e) heat
of binding of other ions such as organic phosphates and
chloride.
In the case of HbA, A H is more exothermic at very alkaline pH values (pH > 9.0) where the Bohr effect is complete
and the contribution of the Bohr protons (endothermic) is
abolished. Hence, as the pH falls, the apparent A H of HbA
reaction becomes less and less exothermic owing to the
310
Table 1. Overall heat of oxygenation of some Arctic ruminant
Hbs in 0.1 M Hepes plus 0.1 M NaCl at pH 7.4 and, for comparison, of human and horse Hbs in the presence of 3mM GriP,.
Ruminant Hbs are modulated in viuo essentially by chloride ions
and not by Grip,. AH values were calculated from the van't Hoff
equation by using the data obtained from 0, equilibria experiments
and corrected for the heat contribution of 0, in solution (-12.5 kJ
mol-I). Confidence limits of the data are 2 5 % .
Species
AH
Reindeer
Musk ox
Cervus
Horse
Man
kJ mol-'
-13.8
- 14.6
-12.9
-28.4
-33.4
LZI
l
1.0-
'
1%
I>
0-
0
-
increasing contribution of the Bohr protons which cancels
some of the heat released upon 0, binding.
A thermodynamic analysis of 0, binding with hemoglobins from different species reveals a series of adaptive mechanisms which are based on the thermodynamic connection
between the binding of heterotropic effectors and the reaction
with oxygen. Hence the 0, binding properties of hemoglobins from Arctic ruminants [l-91, fishes [S-221, high-altitude mammals [23-261, diving animals [27-321, some species of birds [33] and finally human fetus [34] illustrate how
evolution can alter the structural basis of the heterotropic
interactions to optimize the thermodynamics of the oxygenation-deoxygenation cycle in dependence of the physiological
needs of the particular organism.
-1.0-
-2.0
J
Fig. 1. Effect of temperature on 0, equilibria of reindeer hemoglobin measured in 4 % CO,, 0.05 M Tris/HCI pH 7.4 at 10°C
(0),
15°C (0)and 20°C (A). Oxygen pressure (P0J is expressed
in Pa.
Table 2. Apparent heat of oxygenation for musk ox whole blood
and purified hemoglobin at two different pH. The values are corrected for the heat contribution of oxygen in solution (-12.5 kJ
mol-I). In the case of Hb solutions, the experiments were performed
in 0.1 M Tris buffer plus 0.1 M NaC1.
Samples
PH
AH
kJ mol-'
Total blood
7.3
7.6
-13.8
-20.1
Hemoglobin
7.3
7.6
-15.9
-15.9
Mammals and arctic environment
It has long been known that temperature, while it has a
large effect on the position of the 0, dissociation curve of
mammalian hemoglobins, leaves its sigmoidal shape almost
unchanged over a large range of 0, saturation. This observation, together with the exothermic character of the binding
reaction, implies that the came amount of heat is liberated all
along the saturation curve.
It has been recently shown [1 - 81 that hemoglobins from
Arctic and sub-Arctic ruminants (such as reindeer, musk ox
and cervus) under physiological conditions have an overall
oxygenation enthalpy ( A H ) that is much less exothermic than
that reported for human HbA and for other mammalian hemoglobins (Table 1).
The best example of this group is reindeer (Rangifer tarandus) hemoglobin, whose 0, binding is shown as a function
of temperature in Fig. 1. The shape of the 0, binding curve
is markedly temperature-dependent, a phenomenon that is
linked to the unusual temperature independence of the upper
asymptote which represents the high-affinity state (R-state)
of the molecule in the simple two-states allosteric model
[35]. By contrast, the lower asymptote represents the lowaffinity state (T-state) and is strongly exothermic in nature,
much like the effect observed for HbA. This large difference
in the thermodynamics of the two forms of reindeer hemoglobins results in a particular dependence of the temperature
effect on the degree of O2 saturation (Y) of the protein: for
values of Y > 0.6, which are within the range of 0, saturation
at which the protein works in vivo, the overall heat of oxygenation increases from -12.5 kJ mol-' (at Y = 0.6) to al-
most zero as Y tends to 1.0. This result should be considered
with the very low habitat temperature (down to -40°C) experienced by these animals during the year. We suggest that
the physical fitness of these reindeer can in part be attributed
to the unusual thermodynamic properties of their hemoglobins. In fact, as deoxygenation is an endothermic process, in
the peripheral tissues where the temperature may be as much
as 10°C lower than in the lungs and the deep core of the
organism depending on the external conditions, 0, delivery
would be drastically impaired if the molecule were not characterized by a small A H which means that only half as much
heat is needed compared with other mammals.
The same small overall AH of oxygen binding has been
found in the case of hemoglobin from musk ox (Ovibos
muschiatos) an animal which lives in the same Arctic region
(see Table 2). That the Hb molecule from musk ox should
possess the same peculiar features is clearly outlined by
Fig. 2 in which AH values are reported as a function of pH.
It should be recalled that, in the case of human HbA, the
more exothermic value is observed at very alkaline pH values
where the Bohr effect is over and the contribution of the
Bohr protons (endothermic) is abolished. In the case of musk
ox Hb, we have a completely different situation since the
apparent heat of oxygenation is at its maximum (even if
small) value just within the physiological pH range and tends
to zero or even positive values going towards both more acid
and more alkaline pH values. Hence these very small or even
positive A H values are obtained in regions of pH in which
311
\A
7-
-12
E
i
-I
Y
Q
O
12
i
\
7k
7:O
I
a0
PH
Table 3. Overall heat of oxygenation of whale Hb in 0.1 M "rid
HCI plus 0.1 M NaCI, pH 7.4 either with and without (stripped
conditions) 3 mM GriP, and 2 % CO,. A H values were calculated
from the van't Hoff equation by using the data obtained from 0,
equilibria experiments and corrected for the heat contribution of 0,
in solution (-12.5 kJ mol-I). Confidence limits of the data are
56%.
AH
kJ mol-'
Stripped Hb
plus organic phosphates, no CO,
plus organic phosphates and CO,
I
1
7.5
8.0
PH
Fig. 2. Apparent heat of oxygenation for musk ox Hb as a function of pH calculated from the integrated van't Hoff equation.
The values are corrected for the heat contribution of oxygen in solution (-12.5 kJ mol-I). Conditions: 0.1 M Bistris or Tris buffer plus
0.1 M NaCl in the presence of 3 mM Grip,.
Conditions
1
7.0
-64.8
- 23 .O
- 10.4
the alkaline Bohr effect is almost over. We may therefore
exclude a significant involvement of the Bohr protons in determining this unusual AH of oxygen binding and may think
towards either an intrinsic property of the molecule or to the
effect of some other ions whose presence could be important
in vivo in determining the overall function properties of the
Hb from Arctic mammals.
Other examples of adaptive mechanisms resulting from
the interplay of the effects of organic phosphates, carbon dioxide and temperature are shown by the hemoglobin from
the whale Balaenoptera acutorostrata [27 -301. Although
this hemoglobin has a high intrinsic temperature sensitivity,
when the physiologica factors are added to the system, the
overall heat required for the oxygenation falls to - 10.4 kJ
mol-' (Table 3). This feature brings hemoglobin from the
whale into the same category as hemoglobins from Arctic
ruminants. In this respect we have to consider that most of
the whale's body is covered by a thick insulating layer of
blubber but the active muscular parts, like the fins and the
large tail, are not so well insulated, being kept at a lower
temperature by a counter-current heat exchanger to reduce
heat loss. Unloading of oxygen in these active regions of the
whale's body is thus much the same as in the cold leg
muscles of Arctic ruminants, the main difference lying in the
Fig.3. Effect of carbon dioxide: 0, affinity of whale Hb at 20°C
(0,
0 ) and 37°C (A,A) in 0.1 M Tris/HCl plus 0.1 M NaCl in
A) and in the presence (0,A) of 2 % CO,.
the absence (0,
molecular mechanisms used to achieve this low temperature
sensitivity.
Moreover a striking feature of whale hemoglobin is the
temperature dependence of the C0,effect. At 20°C the experimental data follow a trend very similar to human hemoglobin, with a substantial increase of oxygen unloading in the
presence of CO,, but at 37 "C the effect of CO, surprisingly
vanishes over the entire pH range shown in Fig. 3. This can
be explained in terms of the lower temperature encountered
by whale's blood in the fins and tail in comparison with the
rest of the organism. Within the core of the large body, therefore, CO, does not display any allosteric effect because at
37°C the differential binding of this ligand, with respect to
oxy and deoxy structure, is abolished. This allows the hemoglobin to maintain adequate 0, delivery to the other tissues,
where CO, facilitates 0, unloading to power the activity of
the fins and tail at temperatures well below 37°C. The allosteric response to CO, may come into operation once more
in the lungs because of the temperature of the air breathed
by the animal.
In conclusion, the combined effects of organic phosphates, CO, and temperature on hemoglobin in the whale
optimize 0, delivery to all tissues in spite of their relative
heterothennia.
Fish from the Antarctic Ocean
The temperature of the oxygen-rich coastal Antarctic
Ocean is constantly at - 1.87 "C, the equilibrium temperature
of seawater and ice, at which fish from temperate waters
would be unable to survive. In the process of cold adaptation,
Antarctic fish developed unique specializations such as the
well known synthesis of 'antifreeze' (g1yco)peptides which
lower the freezing temperature of blood and other fluids in a
noncolligative way [22]. A further aspect is the modification
of the hematological characteristics, which clearly differentiate Antarctic fish from fish of temperate or tropical climates.
In fact the blood of Antarctic fish contains fewer erythrocytes
and less hemoglobin. This decrease in the number of erythrocytes and hemoglobin content counteracts the temperatureinduced increase of blood viscosity, greatly facilitating the
312
Table 4. Apparent heat of oxygenation of blood or purified hemoglobin components from Antarctic and non-Antarctic fishes.
The values are corrected for the heat contribution of oxygen in solution (-12.5 kJ mol-').
Species
Samples
pH
AH
kJ mol-'
Antarctic fishes
T. newnesi
Hb 1
neutral
alkaline
G. acuticeps
I? hernacchii
N. coriiceps n.
P horchgrevinki
Non-Antarctic fishes
Arapaiama
A ruuna
Musternlus
Srrradmus
A. anguilla
'
Hb
Hb 1
Red cells
Blood
neutral
7.0-8.0
neutral
alkaline
Hb
Hb
Hb
Hb
alkaline
a1kaline
Hb 1
alkaline
alkaline
alkaline
14.6
-10.9
- 2.1
+ 10.0
-
- 8.4
-
14.2
-48.1
-52.7
-51.0
-44.4
-42.7
cardiac work and bringing the energy demand to levels which
the organism is able to tolerate.
As far as the functional properties of hemoglobin is concerned, a thermodynamic analysis of 0, binding has shown
191 that the enthalpy change for oxygenation in the Antarctic
species is very low when compared to fish of temperate
waters (Table 4).In this respect, the behavior of the hemoglobin from two sedentary benthic species i.e. Pagothenia
bernacchii and Gymnodraco acuticeps is very representative
and particularly impressive.
In the former case (Pagothenia bernacchii), in fact, the
large negative Bohr effect is almost temperature-insensitive,
the overall AH of 0, binding being slightly endothermic
(-+I0 kJ/mol 0,j after subtraction of the contribution of
oxygen solubilization [9]. Also in the case of the single hemoglobin from Gymnodraco acuticeps an unusually low cnthalpy change of oxygenation (=-2.0 kJ/mol 0,) has been
clearly observed [9, 221. Morcover, this cold-adapted teleost
is the first fish species in which 0, transport, mediated by a
single hemoglobin, has been found not to be modulated by
pH and allosteric effectors. Although unusual, these features
are in agreement with the general lifestyle of the fish that
being a slow predator does not need a large oxygen turnover.
Hence, in this case, the absence of a Bohr effect appears to
be balanced by the low 0, affinity (Ps0= 4123 Pa at pH 7.0)
of the hemoglobin and by the small amount of energy required during the oxygenation-deoxygenation cycle.
Warm-bodied fish
The strategy of using hemoglobin components with reduced AH values for temperature adaptation was first described in fish and in particular in teleost such as salmon
and trout [13, 361. On the whole it seems that evolutionary
development has favoured a decrease in the temperature sensitivity of 0, affinity of hemoglobin in those species that
have to experience large fluctuations in temperature [14].
A strilung example of such an adaptation can also be
found among some lamnid sharks and tunas which can maintain their bodies at a temperature substantially above (up to
17 "Cj that of the environment 114- 161. This endothermy
is maintained by a counter-current exchange system which
transfers metabolic heat from the veins to the cold blood
7.5
7.0
80
PH
Fig.4. Effect of pH on the oxygen affinity of emperor penguin
(0)and of sea turtle (A) hemoglobins in 0.1 M Hepes plus 0.1 M
NaCl and in the presence of 3 mM InsP, at 37°C (for penguin
Hb) or 3 mM ATP at 20°C (for turtle Hb).
arriving in the arteries from the gills. If hemoglobin in this
case were to bind 0, exothermically, warming of the cold
arterial blood would cause 0, to dissociate and bubble out
of solution with consequent fatal gas emboli.
To solve this problem tuna (Thunnus thynnus) has
evolved a hemoglobin in which the reaction with 0, is endothermic [15,16, 371 as a result of two opposite effects. Thus
in the first two steps of the reaction with 02,AH values are
negative but, for the last two, A H is strongly positive, causing the equilibrium curves at different temperatures to cross
over. It has been suggested that the endothermic nature of
the third and fourth oxygenation steps arises from Bohr proton release [38], but Perutz has pointed out [lo] that it could
derive from four additional hydrogen bonds present in the T
structure that have to be broken during the allosteric transition to the R state.
So far this fascinating endothermic oxygenation and exothermic deoxygenation mechanism displayed by tuna hemoglobin is unique among hemoglobins.
Turtle, penguin and caiman
In order to widen the scope of the emerging scheme we
may have a look at the functional properties of the hemoglobin system from diving vertebrates such as the sea turtle
(Caretta carettaj, the caiman (Caiman crocodylusj and the
emperor penguin (Aptenodytesforsterij. In fact many aspects
of the biology of these animals are distinct enough to suggest
that their respiratory physiology could be particularly interesting.
Turtles, penguins and caimans are fully committed to the
aquatic life being accomplished divers and spending most of
their lives submerged. In this respect, they have developed
particular mechanisms for the maintenance of an adequate
0, supply to tissues under hypoxic conditions.
On the whole, the blood of these animals has to accomplish its 0, transport function under a wide range of con&tions facing marked variations in pH levels and substantial
temperature changes. For example, penguins blood has to
satisfy the 0, demands connected with the extreme life conditions of the Antarctic habitat and with the characteristic
diving behaviour [39].
In the case of both emperor penguin and loggerhead sea
turtle the shape of the Bohr effect seems well adapted for
gas exchange during very prolonged dives [31, 32, 391. In
particular, as far as the Bohr coefficient (dlog P,JdpH at the
313
I
6.5
I
7:O
7.5
J
8.0
PH
Fig. 5. Overall heat of oxygenation for turtle hemoglobin as a
function of pH. AH values were calculated from the integrated van’t
Hoff equation by using the data from 0, equilibria experiments in
0.1 M Hepes plus 0.1 M NaCl and 3 mM ATP. and are corrected for
the heat contribution of 0, in solutions (- 11.5 M mol-’).
mid point of the transition) for 0, binding to turtle hemoglobin is concerned (see Fig. 4j, its amplitude (-0.35) appears
to be 50% smaller, in the presence of physiological allosteric
effector (ATP), than that displayed by human HbA (-0.73)
in the presence of Grip,. Hence the Bohr effect for 0, binding is strongly reduced, showing also a substantial shift of
the mid point of the transition towards acidic pH values (mid
point values are 7.0 and 7.7 for turtle and human hemoglobin
respectively).
On the whole, these findings could be linked to the diving
habit of these animals. In fact, the increase of lactic acid and
the concomitant decrease in pH which should accompany the
prolonged dives of the animals should not affect the 0, affinity preserving their Hbs from a severe and not controlled
stripping of oxygen.
Hence, during diving, 0, delivery from both penguin and
turtle hemoglobins should be modulated essentially by the
partial pressure of 0, at the level of the specific tissue. Moreover, due to the lower A H seen at acid pH (Fig. S), at the
level of flippers, i.e. of those organs which experience a
lower temperature and a great muscular activity, the 0, transport is not impaired allowing the animals to endure more
prolonged periods of anaerobiosis. Hence, through the very
minor enthalpy change observed at acid pH, oxygen delivery
becomes essentially independent of the water temperature the
animal is exposed to during its diving excursions.
Next, in the case of penguins on land in winter, the feet
may also be in close and permanent contact with ice, their
slun temperature being then in the neighbourhood of 0°C.
This observation seems of particular importance with respect
to the reproduction behaviour. In fact, following egg laying,
the incubation period (about 64 days) extends through the
height of the Antarctic winter. During this period the emperor
penguin incubates the egg, holding it on his feet and living
on stored fat reserves. This would result in a significant metabolic acidosis which in turn may be of benefit for tissues
respiration at the level of feet due to the lower A H of oxygen
transport observed at acid pH values ( A H = -10.5 kJ mol
of 0, at pH 6.5). This could allow penguins to maintain their
eggs on their feet without any impairment of oxygen delivery
at this level.
Finally, particular attention should be given to crocodilian hemoglobin since in the red cell its 0, affinity is modulated essentially by carbonate ions as neither organic phosphates nor carbamoyl lower the 0, affinity of Hb and chloride does so only weakly. The complete sequence of the Hb
from caiman [40] shows 102 substitutions with respect to
human hemoglobin. Perutz et al. have clearly shown [41] that
only a few of these substitutions may explain the changes in
allosteric control abolishing or weakening the binding sites
for the usual allosteric effectors and creating a new pair of
binding sites which are complementary to bicarbonate ions
in the deoxy structure (T state) but not in the oxy structure
(R state).
These binding sites formed by Lys EF6(82j and Glu
H22(144) of one P-chain together with the N-terminal residue of its partner chain lie in the cavity between the two
P-chains, where organic phosphates or carbamoyl are bound
in other species. The proposed stereochemical model [41]
shows the N-terminal serine of caiman Hb within exact reach
of the bicarbonate ion so that one of the bicarbonate oxygens
forms a salt bridge with the a-NH: and can also accept a
rather long hydrogen bond from the serine OH. The second
bicarbonate oxygen forms a salt bridge with Lys EF6(82)
and the third oxygen donates a hydrogen bond to one of the
carboxylate oxygens of Glu H22(144j.
It is a pity that the effect of temperature on the functional
properties of this Hb is lacking. In any case, the decrease in
0, affinity brought about by thc interaction of caiman Hb
with bicarbonate ensures that O2 is released from the blood
to the tissues at relatively high partial pressures of this gas.
If the Hb were insensitive to bicarbonate, the venous po2
would be only 931 Pa (compared to 3591 Pa in the presence
of the effect) thereby impairing strongly the flow of oxygen
from the blood to the tissues [42, 431.
Once again the advantage this mechanism gives to the
crocodilians could be related to the diving habit of the animal. In this respect, the simple, direct and reciprocal action
betwecn 0, and carbon dioxide as end product of oxidative
metabolism is suggestive and fascinating.
High-altitude mammals
Mammals living at high altitude are adapted to life under
hypoxic conditions by different mechanisms, as exemplified
by yak and llama hemoglobins. In the Camelidae family the
adaptation of llamas to altitudes as high as SO00 m is obtained by high 0, affinity compared to that of their lowland
relatives of the genus Camelus i.e. Camelus ferus and
Camelus dromedurius [24, 251. The molecular basis of this
effect has been attributed to the B-chains and, in particular,
to the residue at position p(NA2).
Thus, the hemoglobins from both camel species, as
nearly all Hbs from lowland animals, have p chains with His
at position p(NA2). This residue is one of the four amino
acid residues responsible for the binding of Grip, in the
central cavity formed by the p chains in deoxy-Hb. In contrast, all representatives of the genus Lama (Lama glama,
Lama pacos and Lama vicugna) have Asn at position
/32(NA2). Hence, the positively charged histidine in position
P2 in camel is replaced by the neutral asparagine in llama
hemoglobins resulting in a lower binding constant for Grip,
and thus in a increased 0, affinity which is necessary for the
adaptation to life under hypoxic conditions.
A particular case is represented by the hemoglobin from
Lama vicugna which, among llamas, shows the highest O2
314
Table 5. Oxygen tensions at half-saturation (P5,,)
of adult yak
(Bos grunniens), cow (Bos taurus) and llama (Lama vicugna) hemoglobins measured in 0.1 M Hepes plus 0.1 M NaCl and 3 mM
Grip, at 37°C.
Species
Samples
PH
Table 6. Amino acid residues at position a119 and 155 in various
Hbs and their effect on 0, affinity, in 0.1 M Tris or Hepes plus
0.1 M chloride ions at pH 7.2 and 25°C.
Hemoglobin
a119
p55
P,,
HbA
Human mutant I
Human mutant I1
Greylag goose (lowland)
Bar-headed goose (highland)
Pro
Ala
Pro
Pro
Ala
Leu
Leu
Ser
Leu
Leu
760
453
466
373
266
P50
Pa
Pa
Bos grunniens
Hh 1
Hb 2
7.4
1.4
Bos taurus
Hb
7.4
3330
Lama vicugna
Hb
7.4
2211
2664
2398
affinity (Table 5). This is the result of two simultaneous substitutions: that at position p2 His+Asn which reduces the
influence of phosphate as in the other llamas, and that at
position a130(H13) Ala-Thr, which is thought to perturb
the binding of chloride.
The yak (Bos grunnien.y), which belongs to the family
Bovidae, is also a high-altitude animal which is well adapted
to the low 0, partial pressure prevailing in the high mountains of the Himalayas. The adult yak commonly has two
hemoglobins resulting from two types of a chains [26] (few
yaks also have two types of p chains resulting in four hemoglobin phenotypes). These two adult hemoglobins have been
reported to display higher 0, affinity (Table 5 ) with respect
to hemoglobin from cow (Bos taurus) [23]. This functional
difference has been attributed mainly to a single amino acid
substitution, i.e. to the replacement of alanine at position
p135(H13) by valine. This replacement seems to introduce a
bulkier hydrophobic side chain in the vicinity of the heme
that may cause a small change in the H-helix thereby altering
the 0, affinity [ 261.
In both yaks and llamas, it is a pity that the effect of
temperature has not been investigated.
Migratory birds
That the hemoglobin affinity for 0, of animals living at
high altitudes is significantly higher than that of lowland species of similar size [44] is clearly illustrated by the Hbs from
bar-headed and Andean geese [45, 461. The usual habitat of
the Andean goose (Chloephaga melanoptera) is at altitudes
of 5000-6000m in the Andes, whereas the bar-headed
(Anser indicus) goose is subjected to elevations as high as
9200 m, on its migratory flight over the Mount Everest [46]
where the external po2 is only one-third of that at sea level.
Hence the higher O2 affinity displayed by their hemoglobins
may be regarded as a case of adaptation to hypoxia at extreme altitudes thereby helping these birds to exploit ecological niches inaccessible to other species.
The molecular basis of this adaptation is probably evolutionarily significant as it is attributable to a single amino acid
replacement (see Table 6), although this is different in the
two cases, that perturbs the same intersubunit contact (between residues a1 19 and PSS) of the alpl interface, relaxing
the tension of the T structure and raising the 0, affinity of
the molecule. This interpretation has been confirmed by an
elegant piece of work based on protein engineering [46] : two
HbA mutants with substitutions at position a119 and p55 respectively, show a marked increase in 0, affinity (see Table
6) which is even greater than that between the hemoglobins
of the highland and the lowland geese.
\
=
I
9
I
\
L
6.0
1.0
8.0
PH
Fig.6. Overall heat of oxygenation for Hbs from water-hen (W)
and pigeon (A)as a function of pH. AH values were calculated
from the integrated van? Hoff equation by using the data from 0,
equilibria experiments in 0.1 M Bistris or Tris/HCl buffer plus 0.1 M
NaCl and plus 3 mM InsP,. AH values are corrected for the heat
contribution of 0, in solutions (-12.5 kJ mol-I).
It is remarkable that these mutations, although on different globin chains, have occurred at the same intersubunit
contact in two different species of high-flying birds from
widely separated parts of the world. Unfortunately, the
thermodynamics of oxygen binding studied did not consider
the heat that has to be dissipated during flight. In fact, flight
is a very energy-consuming form of locomotion and, as a
result, the metabolic rates of flying birds increase to more
than eight times the resting rate [47, 481. This implies that
during normal sustained flights birds must be able to dissipate more than eight times as much heat as during rest in
order to avoid overheating [47, 481. Considering this particular aspect, a peculiar feature of the hemoglobin from the
water-hen (Galliizula chloropus), a bird capable of prolonged
flight, is the progressive increase of the exothermic character
of 0, binding as the proton concentration increases, as shown
in Fig. 6 [33]. Here the A H of water-hen and pigeon (Columba livia) Hbs is reported as a function of pH. For pigeon
hemoglobin A H is almost independent of pH. The hemoglobin from water-hen behaves quite differently in that AH is at
a minimum (in absolute value) at alkaline pH and tends to
be more exothermic (up to -113 kJ mol-' of O2 at pH 6.3)
315
ence observed in the absence of the effector. Successively on
going from 20' to 37"C, by virtue of the lower overall heat
of oxygenation (AH) displayed by HbF when in the presence
1.5of Grip, (AH = -23 kJ mol-' of 0, for HbF and -36.4 kJ
mol-' for HbA at pH 7.4 and corrected for the heat contribution of 0, in solution), HbA shows a lower 0, affinity than
8
HbF, as it should if 0, has to be transferred from maternal
1.0to fetal blood (see Fig. 7). Hence, the body temperature of
37°C is essential in determining the extent of the difference
0)
0
in oxygen affinity between maternal and fetal blood and then
the amount of oxygen available for the fetus.
Apart from the gas exchange process, we should not dis0.5regard the possibility that the reduced AH observed in fetal
hemoglobin may have some additional physiological meanings since: (a) it may substantially contribute to minimize
the thermal shock that the newborn has to face at birth beI
I
I
I
cause of the sudden change in environment and (b) it may
7.0
7.5
80
have great importance in maintaining the temperature of the
fetus constant by contributing to dissipation of the heat rePH
leased by its metabolic activity. In fact, since more heat is
Fig.7. Effect of pH on the oxygen affinity of human adult (0, absorbed on dissociation of 0, from HbA than is released by
€0)
I,and human fetal (W, 0, 0)
hemoglobins in 0.1 M Hepes 0, binding to HbF, the placenta could be the place where 0,
plus 0.1 M NaCl and in the absence of GriP, at 20°C (0,
O), in
and heat are exchanged in opposite directions.
the presence of 3 mM GriP, at 20°C (a, 0) and 37°C (0,W).
-
2-
I
Conditions: 0.1 M Hepes buffer plus 0.1 M NaCI.
as the pH drops, in spite of the increasing endothermic contribution of the Bohr protons.
These thermodynamic properties seem to accommodate
the problem of heat dissipation that arises when birds have
to fly for a long time. During the activity associated with
prolonged flights, there should be an increased demand for
O,, more hezt produced as a result of the increased rate of
metabolism, and a concomitant decrease in pH brought about
by lactic acid production and/or the increase in temperature.
Hemoglobin reaching the muscles finds a more acid
pH which lowers its 0, affinity and increases its AH of deoxygenation, which in turn helps to cool the whole organism
and to maintain the body temperature at a reasonable level.
Assuming a pH value of about 6.6 in the muscles, upon deoxygenation hemoglobin from water-hen should require at least
three times more heat than HbA, thereby lowering the heat
that has to be dissipated by other means, such as evaporation
of water and convection. It is noteworthy that Columba livia,
like other pigeons, cannot fly for more than 10 min [33, 471
and so its Hb represents an intermediate case.
Fetal human hemoglobin
Although this hemoglobin is representative of a different
situation, it adds useful information for the emerging overall
scheme. HbF is known to display at 20°C a lower affinity
for 0, than HbA when both proteins are in the absence of
organic phosphates [49]. The physiologically important reverse situation is achieved at 37°C upon addition of Grip,
whose lower effect on HbF is related to some amino acid
substitutions present in y chains [50, 511. However, the difference in 0, affinity observed at 37°C is not solely due to
the different modulation power of Grip, with respect to HbA
and HbF. In fact, a reinvestigation, taking into consideration
the different experimental conditions of previous experiments, revealed new aspects once again linked to the interplay of temperature and organic phosphates [34]. In fact,
the lower effect of Grip, on HbF renders the 0, affinity of
the two Hbs almost identical at 20°C abolishing the differ-
CONCLUSIONS
From these examples, it can be seen that the overall
thermodynamics of a biological macromolecule may alter to
cope with special circumstances ; in hemoglobin this is
achieved by linking the basic reaction with the binding of
different ions and effectors whose thermodynamics contribute to the overall effect of temperature.
In this respect whale hemoglobin illustrates nicely how
temperature and heterotropic ligands can cooperate to modulate the basic function and overall thermodynamic characteristics of the protein. In fact, the presence of CO, and organic
phosphates brings about a roughly eightfold decrease in A H
while the temperature controls the regulatory effect of CO,
in switching the differential binding of this ligand on and off.
Through this unusual mechanism, the blood can maintain its
0, concentration around the large body, so meeting the metabolic needs of the fins and huge tail, which between them
have to generate great forward propulsion.
Human fetal and water-hen hemoglobins are examples of
how a protein may tailor its properties in the interests of the
economy of the organism: so the thermodynamic characteristics of these hemoglobins are exploited to ensure that the
heat from metabolism is dissipated, thereby contributing to
maintaining the body temperature constant.
Caiman, goose, yak and llama hemoglobins emphasize
the substantial effect a few (and even a single) amino acid
substitution may have on the regulatory function of a protein
molecule.
The examples reported here outline how a thermodynamic analysis of the reaction with 0, enhances the meaning
of a detailed structural and functional characterization going
deeper into the physiology of the specific organism.
A special thought goes to A. Rossi-Fanelli, E. Antonini, M. Brunori and J. Wyman, the founders of the 'Roman hemoglobin group'.
We wish also to express our gratitude to J. Bonaventura, S. J. Gill
and M. F. Perutz who have supported, over the years, our comparative work by their continuous encouragement and stimulating interest.
316
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