A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans.

Abstract

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(β/δ)(1) and α(1)(β/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.

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A Biochemical−Biophysical Study of Hemoglobins from Woolly
Mammoth, Asian Elephant, and Humans
Yue Yuan,†Tong-Jian Shen,†Priyamvada Gupta,†Nancy T. Ho,†Virgil Simplaceanu,†
Tsuey Chyi S. Tam,†Michael Hofreiter,‡Alan Cooper,§Kevin L. Campbell,⊥and Chien Ho*,†
†Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
‡Department of Biology, University of York, York, YO10 5YW, United Kingdom
§Australian Centre for Ancient DNA, University of Adelaide, Adelaide, SA 5005, Australia
⊥Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
*
S Supporting Information
ABSTRACT: This study is aimed at investigating the molecular basis
of environmental adaptation of woolly mammoth hemoglobin (Hb)
to the harsh thermal conditions of the Pleistocene ice ages. To this
end, we have carried out a comparative biochemical−biophysical
characterization of the structural and functional properties of
recombinant hemoglobins (rHb) from woolly mammoth (rHb
WM) and Asian elephant (rHb AE) in relation to human
hemoglobins Hb A and Hb A2 (a minor component of human
blood). We have obtained oxygen equilibrium curves and calculated
O2affinities, Bohr effects, and the apparent heat of oxygenation (ΔH)
in the presence and absence of allosteric effectors [inorganic
phosphate and inositol hexaphosphate (IHP)]. Here, we show that
the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of
oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP,
suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally,
spectroscopy data indicates that both α1(β/δ)1and α1(β/δ)2interfaces in rHb WM and rHb AE are perturbed, whereas only the
α1δ1interface in Hb A2is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM
presumably facilitated woolly mammoth survival in the Arctic environment.
H
Within the bloodstream, this respiratory protein switches
cyclically between high and low O2-affinity states to control the
delicate balance between the uptake of O2at the alveoli and its
optimal unloading at the tissues. However, because Hb
oxygenation is exothermic, this balance is disrupted by changes
in temperature such that O2 affinity decreases sharply with
increasing temperature and increases with decreasing temper-
ature.1Although this trait is often considered “adaptive” for
enhancing O2 offloading at warm (exercising) muscles, it may
pose considerable challenges for O2delivery to cool extremities
and peripheral tissues of heterothermic mammals and hence is
often countered.2However, the molecular mechanisms by which
the thermal sensitivity of the O2affinity of Hb, which is dictated
by the overall enthalpy of oxygenation (ΔH), has been lowered
through evolution within the various cold-adapted mammalian
lineages are poorly understood.3−5Elephantids are a particularly
good model system to investigate the effects of temperature on
Hb structure−function relationships as they possess both warm-
and cold-adapted members. Specifically, the group evolved in
warm subtemperate climates, where extant Asian and African
lineages are still found, with the ancestors of the extinct woolly
1H NMR
uman normal adult hemoglobin (Hb A) is a hetero-
tetramer consisting of two α-subunits and two β-subunits.1
mammoth (Mammuthus primigenius) abruptly invading high-
latitude environments of Eurasia near the start of the Pleistocene
ice ages some 1.2−2.0 million years ago.6Consistent with this
environmental diaspora, the functional properties of woolly
mammoth Hb were found to differ substantially from those of
Asian elephant Hb, with the most notable change being a
significant reduction in the overall ΔH of the mammoth protein
in the presence of naturally occurring allosteric effector
molecules.6Interestingly, this functional shift comes about despite
the fact that the primary sequences of Asian elephant Hb differ
from those of the mammoth at only one position in the α-globin
chain (K5N) and at three positions (T12A, A86S, and E101Q)
in the β-type globin chain, respectively (Table 1).6Of these
latter residues, β/δ12Ala of the mammoth protein is near the
2,3-bisphosphoglycerate (BPG) binding cleft, β/δ86Ser is in the
heme pocket, and β/δ101Gln is located between 99Asp and
102Asn of the same chain in the intersubunit α1(β/δ)2interface,
which are all critical to the function of Hb.1Indeed, naturally
occurring human mutations at β101 have illustrated that changes
Received:
Revised:
Published: August 2, 2011
May 19, 2011
July 14, 2011
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at this residue position alter both the intrinsic and allosteric
properties of the protein, though the mechanism by which this
residue exerts these effects is still unclear.7
The Hb of elephantids is unusual in that the β-type subunit is
encoded by a chimeric β/δ-fusion gene (HBB/D) that arose via
an unequal crossing over event long before the radiation of this
group.6,8Consequently, the N- and C- terminal halves of the
hybrid elephantid polypeptide are orthologous to the “β” and
“δ” globin chains of other mammals, respectively. Thus, it
would be interesting to probe the structural significance of this
evolutionary change, as the physiochemical properties of
human Hb A and Hb A2(a minor α2δ2tetramer component
of human blood)which differ at only 10 residue positions
(Table 1)9show important differences in regards to
antisickling properties and thermal stability.10
The primary objective of this study is to evaluate and
compare the biochemical factors that alter the temperature
dependence of O2 binding to Asian elephant and mammoth
Hb, with special reference to human Hbs A and A2.
Additionally, we aim to provide new insights into the structural
mechanisms that affect Hb function, thereby aiding the design
of a new generation of medically relevant Hb-based oxygen
carriers (blood substitutes) for use, e.g., during certain
hypothermia-dependent cardiac and brain surgery applica-
tions.11To this end, we obtained the1H NMR spectra of the
four Hbs and measured their oxygen affinity, sensitivity to
various allosteric effectors, and cooperativity at three different
temperatures (37, 29, and 11 °C) and over a range of pH
(pH 8.5 to 5.5).
■MATERIALS AND METHODS
Materials. Hb A and Hb A2were isolated and purified from
human normal blood samples obtained from the local blood
bank using the procedures routine in our laboratory.12,13
Chemicals and restriction enzymes were purchased from major
suppliers, such as Fisher, Sigma, Bio-Rad, Boehringer
Mannheim, New England BioLabs, Pharmacia, Promega, and
United States Biochemicals, Inc., and were used without further
purification.
Construction of Expression Plasmids. The plasmid
(pHE27E) that expresses recombinant Hb AE was constructed
by replacing the human α- and β-globin genes in Hb expression
plasmid pHE212with Asian elephant α-like and β/δ-like
cDNAs. The plasmid (pHE27M) for expressing rHb WM was
constructed by introducing the mammoth-specific residue
mutations (αK5N, β/δT12A, β/δA86S, and β/δE101Q) into
pHE27E using site-directed mutagenesis. The desired muta-
tions were confirmed by DNA sequencing. For additional
details, see ref 6.
Growth and Purification of rHbs. Plasmids for rHb AE
(pHE27E) and rHb WM (pHE27M) were transformed into
E. coli strain JM109, and the cells were grown in a minimal
medium in a 20 L fermentor (B. Braun Biotech International,
Model Biostat C) at 32 °C until the optical density reached
∼10 at 600 nm. Isopropyl β-D-thiogalactopyranoside (IPTG)
was then added at a concentration of 24 mg/L to induce the
expression of the rHbs. After the addition of hemin (25 mg/L),
growth was continued for at least another 4 h. Harvesting was
accomplished through centrifugation. Cell paste was stored at
−80 °C until it was needed. The purification of rHbs followed
Table 1. Amino Acid Residue Differences among the α- and β-Type Globin Chains of Humans Hb A and A2,9Asian Elephant
(rHb AE), and Woolly Mammoth (rHb WM)a
aDifferences from the Hb A sequence are colored in blue. Amino acid residue in rHb WM which differs with that in rHb AE is colored
in red.
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the previous procedure.12,14The purity of the rHbs was then
checked with an electrospray ionization mass spectrometer and
Edman degradation as described previously.12,14All rHbs used
in this study had the correct molecular weights and contained
less than 5% methionine at the amino termini.
Oxygen-Binding Properties of Hbs. Oxygen-equilibrium
curves for these four Hbs were measured in both 0.1 M 2-(N-
morpholino)ethanesulfonic acid (MES) and 0.1 M sodium
phosphate buffers at 11, 29, and 37 °C with a Hemox Analyzer.
Experiments were conducted in the presence and absence of
three-time molar concentrations of inositol hexaphosphate
(IHP), in the pH range 5.5 to 8.5. The sample concentration of
Hbs used 100−120 μM (in terms of heme) was selected to
avoid tetramer−dimer dissociation. Each sample was checked
before and after each measurement for methemoglobin
(met-Hb) in a spectrophotometer. Any sample with greater
than 5% met-Hb was discarded. The partial pressure of O2at
50% Hb saturation (P50), a measure of oxygen affinity, and the
Hill coefficient (n50), a measure of the cooperativity of the
oxygenation process, were calculated for each oxygen-
equilibrium curve. The P50values (mmHg) have an accuracy
of ±5%, and the n50values have an accuracy of ±10%.
1H NMR Spectroscopy. To detect changes in the tertiary
or quaternary structure of the two elephantid Hbs,1H NMR
spectra were obtained on Bruker Avance DRX-300 (results not
shown) and DRX-600 NMR spectrometers. Samples consisted
of aqueous solutions of Hb at a concentration of 5% (3.1 mM
in terms of heme) in 0.1 M sodium phosphate buffer at pH 7.0
in 95% water and 5% deuterium oxide (D2O) and were
assessed at 11, 29, and 37 °C. A jump-and-return pulse
sequence was used to suppress the water signal.1H chemical
shifts were indirectly referenced to the methyl proton
resonance of the sodium salt of 2,2-dimethyl-2-silapentane-5-
sulfonate (DSS) through use of the internal reference of the
water signal at 4.76 ppm downfield of DSS at 29 °C.
■RESULTS
Oxygen-Binding Properties in MES and Phosphate
Buffers. The functional properties of the four Hbs were
studied by measuring their oxygen-binding affinities as a
function of buffer, pH, and temperature in the presence or
absence of IHP (Figure 1). Each of the Hbs studied exhibits
distinct functional properties, with P50 values varying widely
between Hbs. Notably, rHb WM has a lower O2affinity (higher
P50value) than that of rHb AE under the various experimental
conditions (Figure 1). The P50values of Hb A and Hb A2are
very similar and always higher than those of rHb WM and rHb
AE. The P50 values are also affected remarkably by buffer
condition. For instance, in MES buffer and in the absence of
IHP (“stripped” condition), the differences of the P50 values
between rHb WM and rHb AE are very small (Figure 1A) but
become significant in phosphate buffer (Figure 1B) due to their
different response to phosphate ion. Similarly, the addition of
IHP to Hbs significantly decreases the O2 affinities in both
MES buffer and phosphate buffer, but the relative IHP effect is
much stronger in MES buffer. Thus, in the presence of IHP, the
O2affinities of these Hbs are always lower in MES buffer than
in phosphate buffer (Figure 2). The effects of the buffer and
IHP on the O2affinities are also illustrated by the dramatic shift
of the oxygen-binding curves. For example, at pH 7 and 37 °C,
in the absence of IHP, the O2-binding curves for these Hbs are
very close in MES buffer (Figure 3A) but become scattered in
phosphate buffer (Figure 3B). Upon the addition of IHP, all of
the binding curves are shifted to the right in both buffers.
Finally, it is noted that the addition of IHP affects the O2
affinities of Hb A and Hb A2the most, followed by rHb WM,
whereas rHb AE is affected the least by IHP (Figure 3).
The cooperativity of the oxygenation process for all Hbs can
be assessed by the Hill coefficient, n50. The n50values for Hbs
under various experimental conditions are taken as the slope of
the Hill plots at 50% saturation and are summarized in the
Supporting Information Table 1S. The n50 values change
significantly in the presence of IHP but are only slightly affected
by buffer, temperature, and pH. For example, as shown in
Figure 4, the n50 values for both Hb A and Hb A2 are
maintained around the expected value of 2.8−3.0 in the range
of pH 5.8−8.4 in 0.1 M phosphate buffer at 29 °C, while n50
values for rHb WM and rHb AE are found to have lower values
(2.0−2.5) under the same experimental conditions. Upon the
addition of IHP, a broader spectrum of differences in the n50
Figure 1. pH dependence of the oxygen-binding properties (P50, in mmHg) of Hb A (○, red), Hb A2(△, green), rHb WM (◇, blue), and rHb AE
(◻, magenta) in (A) 0.1 M MES buffer and in (B) 0.1 M sodium phosphate buffer in the absence (filled) and presence (open) of IHP and at 37, 29,
and 11 °C.
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Figure 3. Percent of O2saturation of Hb A, Hb A2, rHb Asian elephant (AE), and rHb woolly mammoth (WM) as a function of O2partial pressure
at pH 7.0 and 37 °C in (A) 0.1 M MES buffer and in (B) 0.1 M sodium phosphate (NaPi) buffer in the absence (solid lines) and presence (dashed
lines) of IHP.
Figure 2. Comparison of pH dependence of the oxygen-binding properties [log P50(mmHg)] for (A) Hb A (○, red), (B) rHb WM (◇, blue), and
(C) rHb AE (◻, magenta) measured in 0.1 M MES buffer and in the absence (filled) and presence (open) of IHP and at 11, 29, and 37 °C,
respectively. Measurements conducted for the three Hbs in 0.1 M sodium phosphate buffer (green) are also presented.
Figure 4. Hill coefficient (n50) of Hb A (○, red), Hb A2(△, green), rHb WM (◇, blue), and rHb AE (◻, magenta) as a function of pH at 29 °C
in (A) 0.1 M MES buffer and in (B) 0.1 M sodium phosphate (NaPi) buffer in the absence (filled) and presence (open) of IHP.
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values is observed with n50being generally reduced at lower pH
values and close to n50values measured in the absence of IHP
at higher pHs. Of note, the n50values of rHb WM are generally
lower than those of the other Hbs under all experimental
conditions (Figure 4).
The usual Hb concentration used for our O2-binding measure-
ments is 100−120 μM (in terms of heme). In order to
investigate if rHb WM and rHb AE exhibit unusual tetramer−
dimer dissociation, we have also carried out a concentration-
dependent study of the O2-binding measurements of these two
Hbs together with Hb A over a Hb concentration range of
25−100 μM in 0.1 M sodium phosphate buffer at pH 7.4 and
37 °C. No observable differences in both P50 and n50 values
among these concentrations were found, indicating that there
is no significant tetramer−dimer dissociation over the range of
experimental conditions studied (see Supporting Information
Table 2S).
Bohr Effect. The pH dependence of the oxygen affinity of
Hbs is measured over a range of pH 8.5 to 5.5, and a significant
difference is found in the amplitude of the alkaline Bohr effect
(Δlog P50/ΔpH between pH 6.8 and 8.0) for these Hbs
(Table 2). In the “stripped” condition, the O2-binding affinity
of Hb A shows a stronger Bohr effect (Δlog P50/ΔpH =
−0.53), while rHb WM and rHb AE are characterized by lower
Bohr coefficients (Δlog P50/ΔpH = −0.38 and −0.28,
respectively). However, upon the addition of IHP, Bohr
coefficients of rHb WM and rHb AE reach their maximum
(Δlog P50/ΔpH = −0.81), which is comparable to that of Hb A
(Δlog P50/ΔpH = −0.79). The Bohr effect of rHb WM is
slightly increased (−0.46) in phosphate buffer and is further
increased to about −0.70 upon the addition of IHP, very close
to that of Hb A and A2under the same experimental conditions
(Table 2). However, the Bohr effect of rHb AE remains low
(−0.32) in phosphate buffer and only increase slightly (−0.47)
in the presence of IHP, a value still considerably lower than that
of the other Hbs.
Effect of Temperature on Oxygen Affinity. The
oxygen-binding affinity of Hbs is affected not only by allosteric
effectors and pH but also by temperature.1,15The temperature
dependence of the oxygen affinity of Hbs has been measured at
37, 29, and 11 °C, in the pH range of 5.5−8.5 in MES and
phosphate buffers, respectively, and the results are summarized
in Figure 5 and Supporting Information Figure 1S. As expected,
P50values of Hbs are lower at 11 °C than at 37 °C, indicating
that O2binds tighter to Hbs at lower temperatures (Figure 5).
In the absence of IHP, the P50values of these Hbs are in the
same range, increasing from ∼3 mmHg at 11 °C to ∼16 mmHg
at 37 °C. However, the P50values of rHb WM exhibit a smaller
increase for the same experimental temperature change. As
illustrated in Figure 5A, rHb WM has a lower Δlog P50/ΔT
value (0.023) in MES buffer, compared to Hb A (0.034) and
rHb AE (0.028), where ΔT represents the difference bet-
ween two temperatures (in °C) and Δlog P50is the difference
in P50(mmHg) at the two temperatures. In phosphate buffer,
the Δlog P50/ΔT values for these Hbs are all reduced, through
that of rHb WM (0.019) remains consistently lower than that
of Hb A (0.023) and rHb AE (0.024). Upon the addition of
IHP, all of the P50 values at each experimental temperature
increase, but the absolute values vary for each Hb because of
their different response to IHP. In order to illustrate the
temperature effect more clearly, the apparent heat of oxygen-
ation, ΔH, is calculated from the van’t Hoff eq 1 on the basis of
Δlog P50at the two different temperatures (T1and T2, in K):
(1)
ΔH includes the heat of oxygen solvation (−3 kcal mol−1).
The results of the exothermic oxygenation enthalpy (ΔH, kJ
mol−1, where 1 cal = 4.184 J) presented in Table 3 have been
corrected for this value. It is noted that the ΔH values for the
four Hbs are all pH dependent (Figure 6). For example, in
phosphate buffer, the ΔH value for rHb WM changes from
Table 2. Alkaline Bohr Effect (Δlog P50/ΔpH) of
Hemoglobins from Human (Hb A and Hb A2), Woolly
Mammoth (rHb WM), and Asian Elephant (rHb AE) in the
Presence and Absence of Inositol Hexaphosphate (IHP)a
conditionHb A
−0.53
−0.79
−0.45
−0.67
Hb A2
rHb WM
−0.38
−0.81
−0.46
−0.72
rHb AE
−0.28
−0.81
−0.32
−0.47
MES buffer
−IHP
+IHP
−IHP
+IHP
NaPi buffer
−0.43
−0.71
aMeasurements were conducted at 37 °C and over the pH range 7.0−
7.8 in 0.1 M MES and sodium phosphate (NaPi) buffers at a
hemoglobin concentration of 100 μM.
Figure 5. Comparison of temperature dependence of the oxygen-binding properties [log P50(mmHg)] in the absence (filled) and presence (open)
of IHP for Hb A (○, red), Hb A2(△, green), rHb WM (◇, blue), and rHb AE (◻, magenta) measured at 11, 29, and 37 °C: (A) at pH 7.0 in
0.1 M MES buffer; (B) at pH 7.4 in 0.1 M sodium phosphate (NaPi) buffer.
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−41.0 kJ mol−1at pH 5.8 to −18.7 kJ mol−1at pH 7.4. At pH
7.0 in the “stripped” condition, the ΔH values of rHb WM and
rHb AE are −25.9 and −30.6 kJ mol−1, respectively, much
lower (in absolute terms) than that of human Hb A (−46.1 kJ
mol−1); i.e., rHb WM and rHb AE show a smaller temperature
effect than Hb A. In phosphate buffer, the ΔH values of rHb
WM are less negative than those of rHb AE, while the ΔH
values of Hb A and Hb A2are very similar over the range of
experimental conditions. When IHP is present, the ΔH values
of the four Hbs are all significantly decreased (in absolute
terms), with that of rHb WM remaining slightly lower than that
of rHb AE. For all four Hbs, the lowest ΔH values (in absolute
terms) are observed when both phosphate and IHP are present
as functional modulators, suggesting an additive effect of
phosphate and IHP to the ΔH value.
1H NMR Spectra of Hemoglobins.
were recorded for the Hbs in the deoxy and the CO forms in
the absence and presence of IHP at 11, 29, and 37 °C. The
exchangeable proton resonances of α103His and α122His of
the four Hbs studied in the CO and deoxy forms are shown in
Figure 7. In Hb A, the resonance at 12.2 ppm has been assigned
to the side chain Nε2H group of α103His,16−19and the
resonance at 12.9 ppm has been assigned to the side chain
Nε2H group of α122His.18In the CO form of Hbs (Figure
7A), the peak at 12.2 ppm (α103His) is not present in the
spectra of rHb AE or rHb WM but appears in the spectra for
Hb A and A2. Additionally, the peak at 12.9 ppm (α122His) in
Hb A is shifted downfield in the spectra of rHb AE and rHb
WM (which have α- and β/δ-chains) and Hb A2(which has
α- and δ-chains). In the spectrum of deoxy-Hb A (Figure 7C),
the resonance at 14 ppm was assigned to the H-bond between
α42Tyr and β99Asp and has been used as a T-state marker at
the α1β2 interface.20In the presence of IHP, the T-marker
starts to appear in the spectra of the CO form of Hb A and Hb
A2at a lower temperature (11 °C) but is not seen in the spectra
of the CO form of rHb AE and rHb WM (Figure 7B).
Significant differences are observed in the deoxy state of the
Hbs (Figure 7C). As reported previously, the T-marker in
deoxy-Hb A2is present at the same spectral position as that of
deoxy-Hb A.13These T-state markers shift 0.1−0.3 ppm
downfield in the spectra of rHb WM and rHb AE, and the peak
at 11.1 ppm assigned to β37Trp also shifts 0.2 ppm downfield
at all temperatures, indicating a perturbation of the α1(β/δ)2
interface in rHb WM and rHb AE (Figure 7C).
1H NMR spectra
Table 3. Apparent Enthalpy of Oxygenation (ΔH; kJ mol−1
O2) Values of Human (Hb A and Hb A2), Woolly Mammoth
(rHb WM), and Asian Elephant (rHb AE) Hemoglobin as a
Function of pHa
ΔH (kJ mol−1)
buffer pHHb A
−42.0
−38.7
−40.8
−46.1
−42.7
−30.4
−19.9
−18.3
−19.4
−24.9
−27.1
−30.4
−41.4
−39.2
−33.0
−27.5
−24.3
−32.4
−29.5
−27.1
−28.3
Hb A2
rHb
WM
−29.1
rHb AE
−30.9MES 5.80
6.19
6.53
7.00
7.40
7.88
5.80
6.19
6.53
7.00
7.40
7.88
5.79
6.23
6.57
7.04
7.44
7.82
5.80
6.26
6.84
7.06
7.42
7.81
8.03
−28.3
−25.9
−20.4
−30.6
−26.7
−31.5
−31.7
−31.1MES + IHP
−28.1
−19.1
−24.6
−22.2
−14.6
−41.0
−36.7
−17.5
−42.5
−41.6
NaPi
−44.5
−42.7
−28.7
−26.8
−33.7
−35.9
−32.2
−21.9
−18.7
−21.0
−38.8
−32.2
−23.7
−33.1
−30.6
−32.8
−40.9
−38.2
−26.0
−11.8
−15.9
−24.0
−40.9
−35
−31.5
NaPi + IHP
−31.3
−17.4
−22.4
−10.7
−20.0
−25.4
−10.4
−10.7
−28.4
−35
−27
“stripped”6
“stripped” + 0.1 M
Cl−6
0.1 M Cl−+ 2.5 M
BPG6
aMean ΔH values of Hbs were calculated from P50values measured
in the absence and presence of inositol hexaphosphate (IHP) in
0.1 M MES and sodium phosphate (NaPi) buffers at 11, 29, and
37 °C. Confidence limits of ΔH values are ±15%. Mean ΔH values
previously determined for woolly mammoth and Asian elephant
rHbs in 0.1 M HEPES buffer at pH 7.0 and 7.4 and over the
temperature ranges 10 and 25 °C and 25 and 37 °C6are presented for
comparison.
−41.0
−19.3
−28.1
Figure 6. Apparent enthalpy of oxygenation (ΔH; kJ mol−1O2) values of Hb A (○, red), Hb A2(△, green), rHb WM (◇, blue), and rHb AE (◻,
magenta), as a function of pH. Mean ΔH values of Hbs were calculated from P50values measured at 11, 29, and 37 °C (A) in 0.1 M MES buffer and
(B) in 0.1 M sodium phosphate (NaPi) buffer and in the absence (filled) and presence (open) of IHP. Confidence limits of ΔH values are ±15%.
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Ring-current-shifted resonances at −1.5 to −2.0 ppm (Figure
7D) provide information about the tertiary structure around
the heme pocket.16The resonances at −1.75 and −1.82 ppm
have been assigned to the γ2-CH3 group of E11Val of the
α- and β-chains of HbCO A, respectively.21,22For rHb WM and
rHb AE, the E11Val methyl resonances of both the α- and
β/δ-chains are shifted upfield to −2.01 ppm, while the
resonances of Hb A2are not affected.
■DISCUSSION
Response to IHP. Mammals can be divided into two
groups based on how the O2-binding affinities of their Hbs
respond to allosteric effectors. The O2-binding affinities of Hbs
from humans, pigs, dogs, and most primates are decreased in
the presence of organic phosphate, whereas those from cats and
ruminants are not sensitive to organic phosphate but exhibit
significant effects in the presence of chloride ions.23−25Our
study has examined the properties of rHb WM and rHb AE
with respect to their interactions with phosphate and IHP. Our
results have confirmed that rHb WM and rHb AE belong to the
group of mammalian Hbs that exhibit an intrinsically high
oxygen affinity, which can be modulated by phosphate anions
(i.e., phosphate and IHP). Comparisons between the P50values
obtained in MES and sodium phosphate buffers show that the
O2-binding affinities of Hb A, rHb WM, and rHb AE signifi-
cantly decrease in phosphate buffer and/or in the presence of
IHP (Figure 2). However, the IHP effect is stronger in MES
buffer than in phosphate buffer, suggesting that the allosteric
effects of phosphate and IHP are not synergistic with respect to
the O2binding. Conversely, our results indicate that these two
anions compete for the same binding sites. Thus, the changes of
P50values of these Hbs upon the addition of IHP reach their
maximum in MES buffer and cannot be increased further in
phosphate buffer.
On the basis of our current studies, rHb WM exhibits a
stronger response to allosteric effectors (Hþ, phosphate, and
IHP), than rHb AE, despite possessing only three amino acid
differences in their β/δ-chain sequences.6The functional
properties of β/δ101Gln in rHb WM and β/δ101Glu in rHb
AE are of particular interest because this residue is located in
the α1(β/δ)2contact region. In liganded Hb A, β101Glu is in
close contact with α94 residue, while in the deoxy-Hb A, it has
contacts with both β94Asp and α96Val residues.1,26Thus, this
residue is involved in the stabilization of both unliganded and
liganded tetramers of Hbs. Various mutations of Hb A at
β101Glu have been studied, but the role of this residue is still
not clear.7All mutant Hbs at this position have an elevated O2
affinity relative to Hb A, except for the mutant Hb Rush
(β101Glu→Gln), which is the only β101 mutant having a
higher sensitivity to chloride ion than Hb A. Consequently,
despite possessing a high intrinsic O2affinity (as in the other
β101 mutants), the O2affinity of Hb Rush is decreased to a
slightly lower value than that of Hb A in the presence of 0.1 M
chloride.7As suggested by Campbell et al.,6the β/δ101Glu→
Gln substitution in rHb WM might play the same role as in Hb
A, which could allow the positive charge of β/δ104Arg to form
an additional Cl−binding site within the α1(β/δ)2interface of
the deoxy-state molecule. This proposed additional chloride-
binding site in rHb WM might also be responsible for the
increased allosteric effect caused by the addition of phosphate
and IHP, as shown by the drastic decrease in O2affinity of rHb
WM in the presence of these allosteric effectors.
Bohr Effect. Oxygen-binding experiments show that both
inorganic phosphate and IHP affect the functional behavior of
rHb WM and rHb AE, not limited only to the absolute value of
the oxygen affinity, but also affecting the amplitude of the Bohr
effect (Δlog P50/ΔpH). As shown in Table 2, in the “stripped”
condition (i.e., MES buffer and in the absence of IHP), the
Δlog P50/ΔpH values of rHb WM and rHb AE are lower
(in absolute terms) than that of Hb A between pH 6.8 and 8.0.
Bohr effects of the two elephantid rHbs are slightly influenced
by the presence of inorganic phosphate but are markedly
affected by the presence of IHP. For Hb A, it is known that
there are a number of amino acid residues that contribute to the
observed Bohr effects, including the N-terminal residues27,28
and a large number of surface His residues.29−32In 0.1 M
HEPES buffer with 0.1 M chloride, β146His contributes the
most to the alkaline Bohr effect (63% at pH 7.4) among those
surface histidyl residues, while β143His contributes the most to
the acid Bohr effect (71% at pH 5.1).29−32These two histidyl
residues are also present in rHb WM and rHb AE and
presumably also contribute to the Bohr effect of these proteins.
It is noted that the number of histidyl residues in rHb WM and
rHb AE is the same as in Hb A, but the locations of these
residues are different (Table 1). β2His and β116His in Hb A,
which exert moderate negative and moderate positive
contributions, repectively, to the Bohr effect,31,32have been
replaced by β/δ2Asn and β/δ116Arg in rHb WM and rHb AE.
Thus, the contributions from these two histidyl residues to the
Bohr effect are absent in rHb WM and rHb AE. Consequently,
Figure 7.1H NMR spectra (600 MHz) of rHbs in 95% H2O, 5%
D2O, and 0.1 M sodium phosphate buffer. Exchangeable proton
resonances at pH 7.0 and 11, 29, and 37 °C, in the CO form in the
absence (A) and presence (B) of IHP and in the deoxy form (C) are
presented. (D) Ring-current-shifted resonances of the CO form of
Hbs and rHbs at pH 7.0 and 29 °C.
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the low Bohr effects observed for these two Hbs (Table 2) may
arise from substitutions at β/δ44(Ser→His) and β/δ56(Gly→
His), which presumably contribute negatively to the Bohr effect
of these two proteins. Although rHb AE has the identical
histidyl residues as rHb WM, it exhibits a weaker Bohr effect in
phosphate buffer, even in the presence of IHP, suggesting that
the contributions from the histidyl residues might not account
for all of the Bohr effect of these two rHbs. The lower Bohr
effect of rHb AE is consistent with its weaker response to
phosphate and IHP relative to rHb WM (Table 2).
Temperature Effects. The oxygenation of Hb is exother-
mic, with increasing the temperature lowering the O2affinity
directly by weakening the hydrogen bonds between Hb and
O2.1,33,34Arctic ruminants (e.g., musk ox and reindeer) are
routinely subjected to extremely low environmental temper-
atures. Hence, to reduce the heat loss under these conditions,
these animals exploit countercurrent heat exchangers in the
extremities that allow them to maintain markedly lower tissue
temperatures at these sites.3Thus, a decrease in the tem-
perature sensitivity of oxygen binding could be a functional
strategy which allows unloading O2 to the cool peripheral
tissues. This phenomenon has been observed in the previous
studies for the Hbs of a number of subarctic and arctic
mammals, including Eskimo dog, musk ox, and reindeer.5,35,36
The apparent heat of oxygenation, ΔH, is used to evaluate the
temperature effect on the O2affinities of Hbs. On the basis of
the ΔH values, the Hbs of various species can be broadly
divided into two groups. The Hbs of the first group, including
reindeer Hb and musk ox Hb, have intrinsically low ΔH values
even in the absence of allosteric effectors.5The second group of
Hbs can be further divided based on whether the large negative
ΔH of these Hbs becomes less negative in the presence of
2,3-bisphosphoglyceric acid (2,3-BPG), such as pig Hb and
fetal human Hb (Hb F), or whether the ΔH is not very
sensitive to the addition of 2,3-BPG, such as Hb A.5In the
“stripped” condition, rHb WM and rHb AE have a less negative
ΔH of oxygenation when compared with Hb A (Table 3 and
Figure 6A). In phosphate buffer, the ΔH values of rHb WM
and rHb AE decrease slightly and remain in the same range as
in MES buffer, while that of Hb A dramatically decrease
(in absolute terms) compared to that in MES buffer (Table 3
and Figure 6B). Further comparisons between rHb WM and
rHb AE show that although similar ΔH values are observed in
MES buffer, rHb WM has much less negative ΔH values in
phosphate buffer (Figure 6B), which can be attributed to the
fact that the O2affinity of rHb WM has a stronger response to
phosphate, a weak allosteric effector. When IHP is present, the
ΔH values of Hbs are greatly affected as the result of a change
in the P50values. Differences between the ΔH values of the
various Hbs remain but become smaller due to the strong
allosteric effect of IHP (Figure 6). The less negative values of
the ΔH values observed upon the presence of phosphate and/or
IHP suggest that the apparent temperature effect is dependent
on the ability of each of the Hbs to respond to the allosteric
effectors. On the basis of the previous studies on the Hbs of
various arctic animals,5a potential structural explanation has
been proposed, suggesting that the amino acid residues at β8,
β76, and β77 positions form an “additional” chloride-binding
site (relative to Hb A), which is responsible for lowering ΔH of
these proteins.37,38Additionally, the mutation of β76Ala→Lys
might be responsible for the synergistic effect of 2,3-BPG and
chloride on the O2affinity of these Hbs.38In the Hbs of arctic
animals, such as reindeer and ox, the amino acid residue at β76
is Lys, and the ΔH values of these Hbs are less negative than
that of horse Hb, which has Ala at β76. Both rHb WM and rHb
AE have β/δ76Lys, the same as in reindeer Hb. Our previous
study of rHb WM suggests the E101Q substitution
in rHb WM creates an additional binding site for allosteric
effectors, which contributes to the less negative ΔH of
oxygenation relative to rHb AE, thus forming part of the
physiological adaptation of woolly mammoth.6The less negative
ΔH values of rHb WM observed in the present work confirm
the lower temperature effect of rHb WM compared to that of
rHb AE.6
On the basis of our measurements, the ΔH values of rHb
WM are in the same range as those of Hbs from other arctic
animals obtained under similar experimental conditions.5For
example, in 0.1 M HEPES buffer with 0.1 M NaCl in the
presence of 2,3-BPG, the ΔH of reindeer Hb is −14.0 kJ
mol−1,5whereas the values calculated for rHb WM are −18.7 kJ
mol−1in 0.1 M sodium phosphate buffer (current result) and
−19.3 kJ mol−1in the presence of Cl−and BPG.6These low
ΔH values of rHb WM are consistent with life in the arctic
environment since the O2affinity of its Hb is less affected by
low temperature, and therefore, it would be easier to unload O2
to the cool peripheral tissues.6The ΔH value of rHb AE is
−30.6 kJ mol−1in phosphate buffer, which is more negative
than that of rHb WM. However, it is reduced by the addition of
IHP (−15.9 kJ mol−1), a value only slightly higher than that of
rHb WM (−10.4 kJ mol−1). This suggests that a lower ΔH
value observed from rHb AE might be attributed to the
stronger allosteric effect of IHP and that the reduction in the
ΔH of rHb AE is less pronounced in the presence of 2,3-BPG.6
In the stripped condition, the ΔH value for Hb A (−46.1 kJ
mol−1at pH 7.0) is more negative than that of rHb WM
(−25.9 kJ mol−1), and it is only slightly increased by 2,3-BPG
in the presence of chloride ions.5,39In our study, when Hbs are
saturated with IHP, the ΔH values for Hb A are significantly
increased to the same range as that of rHb WM and rHb AE,
suggesting that the O2affinity and the ΔH value of Hbs are
dependent on the property and the concentration of the
allosteric effectors. It could also be the case for Hbs in the
blood cells. For example, Asian and African elephant blood
have slightly different functional properties, with the O2affinity
of Asian elephant blood being lower than that of African
elephant due to an increased 2,3-BPG concentration in the red
cells.40,41
Structural Information from
NMR spectra of Hbs in 0.1 M sodium phosphate buffer at
pH 7.0 were assessed at 11, 29, and 37 °C. One significant
change shown in the1H NMR spectra is that the peak at
12.2 ppm disappears in the CO and deoxy forms of rHb WM
and rHb AE (Figure 7). In Hb A, this resonance has been
assigned to the side chain Nε2H group of α103His, which is
hydrogen-bonded to β131Gln.16−19The resonance at 12.9 ppm
has been assigned to the side chain Nε2H group of α122His,18
which forms a water-mediated H-bond with the side chain of
β35Tyr. Both H-bonds are located at the α1β1-subunit interface
of Hb A. Our previous study of the mutant rHb (β131Gln→
Glu) shows that the peak at 12.2 ppm in the
spectrum disappears, suggesting that the H-bond between
α103His and β131Gln does not exist due to the replacement at
the β131 position.42In rHb WM and rHb AE, the amino acid
residue of α103His is the same as that in Hb A, but β131Gln
existing in Hb A is changed to β/δ131Glu (Table 1). Thus, the
1H NMR Studies.
1H
1H NMR
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disappearance of the resonance at 12.2 ppm in the1H NMR
spectra of rHb WM and rHb AE is likely to be caused by this
mutation, as reported for the mutant rHb (β131Gln→Glu).42
Our previous studies for rHb (β131Gln→Glu) also show a
slight downfield shift of the α122His resonance to 13.1 ppm,
indicating that the perturbations caused by the replacement at
β131 are not just confined to the mutation site but also affect
the environment of α122His.42This also could be true for the
case of rHb WM and rHb AE. Thus, the changes in1H NMR
spectra on the interface histidyl resonances of α103His and
α122His could be attributed to the replacement of β131Gln→
Glu in rHb WM and rHb AE. In the deoxy state of rHb WM
and rHb AE (Figure 7C), the T-state markers located at
14.2 ppm and the peak of β37Trp at 11.1 ppm are shifted
downfield, indicating a perturbation of the α1(β/δ)2interface
as well (Figure 7). Thus, in rHb AE and rHb WM, both
α1(β/δ)1 and α1(β/δ)2 interfaces are perturbed in both the
deoxy and CO forms in the absence and presence of IHP as
compared to those of Hb A and Hb A2. From the previous
functional studies of the mutant rHb (β131Gln→Glu), it was
shown that this mutation has only a small impact on the oxygen
affinity of the Hb molecule.42Thus, although a significant
change is observed in the1H NMR spectra, the replacement of
β131Gln→Glu might not have an important effect on the
function of rHb WM and rHb AE.
On the basis of the previous studies of Hb A, the non-
exchangeable ring current-shifted proton resonances at −1.75
and −1.82 ppm have been assigned to the γ2-CH3group of the
α62Val and β67Val residues of HbCO A, respectively.15,20,21
These resonances provide information about the geometry/
environment of the heme pocket. The ring-current-shifted
resonances for these two valyl residues are resolved in the CO
form of Hb A and Hb A2, but both shift upfield to −2.0 ppm in
the CO form of rHb WM and rHb AE (Figure 7D). These
results suggest that the distal heme pockets of rHb WM and
rHb AE are altered as compared to those of Hb A and Hb A2.
Comparing the amino acid sequences of these Hbs, several
replacements in rHb WM and rHb AE can be found near
α62Val and β67Val, including α63Ala→Gly, α64Asp→Glu,
β65Lys→Glu, and β69Gly→Thr. Previous studies have
indicated that the mutations in the heme pocket of Hb A can
change the O2affinity by affecting the tertiary structure of the
protein.34,43,44Our NMR studies reported here provide direct
evidence showing that the geometry/environment of the heme
pockets of rHb WM and rHb AE is different with respect to
those of human Hbs and that these changes may be related to
the higher O2-binding affinity of these Hbs.
Structural Information from Hb A2. Hb A2 exhibits a
remarkable structural similarity to Hb A. The α-chains are the
same in these two Hbs, and there are only 10 amino acid
substitutions in the δ-chain of Hb A2compared to the β-chain
of Hb A. On the basis of the ring-current-shifted resonances of
E11Val, no significant change in the distal heme pocket was
detected due to the δ-chain replacement (Figure 7D). In the1H
NMR spectra of Hb A2, the resonance at 13.1 ppm shows a
slight downfield shift, similar to that of rHb WM and rHb AE,
while the resonance at 12.2 ppm is unchanged, suggesting that
the δ-chain replacement in Hb A2conserves the αlδ2interface,
but only slightly perturbs the αlδ1interface.13The perturbation
on the intradimer interface is shown more clearly in the1H
NMR spectra of rHb WM and rHb AE due to the combination
effect of the β/δ-chain replacement and the mutation of
β/δ131Gln→Glu.
Perutz and Raidt reported that Hb A2 was more resistant
to thermal denaturation than Hb A.45They suggested that two
amino acid residues in the helices G (δ116Arg) and H
(δ126Met) of Hb A2 might be responsible for its higher
thermal stability. It is speculated that δ116Arg could make
an extra hydrogen bond at the α1δ1interface and that δ126Met
(H4) could make intrasubunit nonpolar contacts with δ11Val
of helix A.45However, recent X-ray crystal studies of Hb A2did
not support these additional interactions, but nonpolar contacts
formed between helices A and H were found, which
presumably contribute to the higher thermal stability of Hb
A2.10,46Notably, rHb WM and rHb AE also possess Arg at
position 116 of their β-type chains (Table 1), which may
similarly elevate the thermal stability of these two Hbs. The
X-ray structural studies also suggest that the higher O2-binding
affinity of Hb A2 may result from the slighter larger α1δ2
interface of Hb A2and an additional hydrogen bond at the α1δ2
(or α2δ1) interface between α94Asp and δ37Trp in Hb A2.10
This could also be true for rHb WM and rHb AE.
■CONCLUSION
It is important to understand how the oxygen transport of Hbs
is controlled by the combined action of temperature and
ligands. Our studies on rHb WM and rHb AE have confirmed
that these two Hbs respond differently to changes in experi-
mental conditions. In general, the O2affinity of rHb WM has a
larger response to allosteric effectors and is less sensitive to
temperature change than rHb AE. These features are related to
its structure and appear to arise from at least two of the three
mutations found on the β/δ chain of this extinct species. It
should be noted that our biochemical−biophysical study is
limited to only the Hb molecule. In red cells, the factors that
affect the oxygen affinity are more complex. Thus, the negative
influences, such as a decrease in oxygen unloading due to a
lower temperature, may be compensated by the action of one
or more other factors. For example, it has been shown that
blood of African and Asian elephants possess slightly different
functional properties, with the O2 affinity of Asian elephant
blood being lower than that of African elephant blood due to
an increased 2,3-BPG concentration in the red cells.40,41It is
possible that such subtle difference also existed in woolly
mammoth blood. The distinct structural features of rHb WM
provide a part of the basis for woolly mammoth survival in
the arctic environment. Further investigations are needed
for applying these structural features to the design of a new
generation of medically relevant Hb-based oxygen carriers.
■ASSOCIATED CONTENT
*
Table 1S showing the Hill coefficient (n50); Table 2S and
Figure 1S showing the concentration and temperature
dependence of the oxygen-binding properties (P50) of Hbs
under various experimental conditions, respectively. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■AUTHOR INFORMATION
Corresponding Author
*Phone: 412-268-3395. Fax: 412-268-7083. E-mail: chienho@
andrew.cmu.edu.
S Supporting Information
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Funding
This work is supported by a research grant from the National
Institute of Health (R01GM084614).
■ACKNOWLEDGMENTS
We thank Dr. E. Ann Pratt for helpful comments on our
manuscript.
■ABBREVIATIONS
Hb A, human normal adult hemoglobin, the α2β2tetramer; Hb
A2, a minor component of human normal adult hemoglobin,
the α2δ2 tetramer; rHb, recombinant Hb; rHb WM,
recombinant woolly mammoth Hb; rHb AE, recombinant
Asian elephant Hb; HbCO, carbonmonoxyhemoglobin; deoxy-
Hb, deoxyhemoglobin; met-Hb, methemoglobin; NMR,
nuclear magnetic resonance; DSS, 2,2-dimethyl-2-silapentane-
5-sulfonate; MES, 2-(N-morpholino)ethanesulfonic acid; 2,3-
BPG, 2,3-bisphosphoglycerate; IHP, inositol hexaphosphate.
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