The interaction of inositol hexakisphosphate
(IHP) with oxygenated human adult hemoglobin (Hb) was investigated at
25 °C. The affinity of IHP for oxygenated Hb is strongly
pH-dependent, and potentiometric measurements of proton
uptake and release upon IHP addition have shown that over the range
between pH 8.0 and pH 6.0 in oxygenated Hb there are three groups of
residues that change their pKa values after IHP
addition, likely because of their interaction with negative charges of
the heterotropic effector. On the basis of previous calculations on the
electrostatic properties of human Hb (Matthew, J. B., Hanania,
G. I. H., and Gurd, F. R. N. (1979)
Biochemistry 18, 1919-1928; Lee, A. W.-m., Karplus,
M., Poyart, C., and Bursaux, E. (1988) Biochemistry 27, 1285-1301), two of these groups might be Val1
and
His143
, which are located in the
1
2 dyad axis, where they have been also
proposed to interact with 2,3-diphosphoglycerate, whereas the third
group does not appear easily identifiable. Calorimetric measurements of
the heat associated with IHP binding at different pH values over the
same range indicate that IHP binding is mostly enthalpy-driven at
pH < 7 and mostly entropy-driven at pH > 7.
 |
INTRODUCTION |
Human hemoglobin (Hb)1
is functionally modulated by several non-heme ligands, such as organic
phosphates (i.e. 2,3-diphosphoglycerate (2,3-DPG) and
myo-inositol hexakisphosphate (IHP)), protons, and chloride
ions (1-5), which bind at heterotropic interaction sites, topologically distinct from the heme at which homotropic ligands bind.
The structure of this binding pocket has been determined for the
interaction of 2,3-DPG (6), which has been shown to bind at the
interface between the two
-chains, mainly involving three residues
from either one of
-subunits (i.e. HisNA2(
2),
LysEF6(
82), and HisH21(
143), see Ref. 6).
In more recent years, another organic phosphate, namely IHP (closely
related to the inositol pentaphosphate, which is the physiological
effector in avian erythrocytes; see Ref. 7), has often been employed to
study the modulation of functional properties of human Hb (8, 9). It
possesses additional negative charges with respect to 2,3-DPG, and it
displays a much larger effect, which suggests the occurrence of
additional electrostatic interactions with respect to 2,3-DPG, as from
early model building studies on deoxy Hb (10). Therefore, the enhanced
functional effect of IHP on the O2 binding properties of
human Hb with respect to 2,3-DPG could be related to a more widespread
interaction surface, with the possibility of modulating ligand-linked
conformational changes taking place over a larger portion of the whole
tetramer.
However, a comprehension of the origin for this enhanced effect starts
from the characterization of the IHP interaction energy with deoxyHb
and with oxyHb. Previous studies have shown that IHP binds
HbO2, and its binding properties are
pH-dependent (11, 12). In this study, we have carried out a
detailed analysis of the interaction of IHP with human
HbO2, measuring the effect on (a) proton
titration, (b) O2 dissociation kinetics from
fully liganded tetramer, and (c) heat associated to the
reaction in order to give a quantitative description of the system and
of the interplay between IHP and proton interaction with human
HbO2.
 |
EXPERIMENTAL PROCEDURES |
Human HbO2 was obtained from the blood of healthy
volunteers and stripped of anions according to the procedure reported
by Riggs (13). Cells were washed three times with iso-osmotic NaCl solutions by centrifugation at 1000 × g, and packed
cells were lysed by adding 2 volumes of cold bidistilled water. Stroma
were removed by centrifugation at 12,000 × g for 30 min. Hemolysate was first filtered through a Sephadex G-25 column,
equilibrated with 0.01 M Tris/HCl buffer, pH 8.0, and EDTA
10
5 M, and afterward it was passed through a
column of mixed bed ion-exchange resin (Bio-Rad AG501-X8). For proton
titration experiments, Hb solution was concentrated on Amicon YM10
(Bio-Rad) membranes. For all other experiments, the sample was then
extensively dialyzed versus the desired buffer. All
experiments were performed at 25 °C in the presence of 0.1 M NaCl.
Titrations were performed at 25 °C using a thermostatted
autotitrator (Radiometer, Copenhagen, Denmark) equipped with a SAM90 sample station, ABU93 triburette unity, and VIT90 titration controller, adding automated 100-µl aliquots of 2 mM NaOH (prepared
from 0.01 M Normex and checked by acid titration). For each
experiment, three solutions (between 0.75 and 2.00 ml) were titrated,
namely (a) HbO2 ranging between 1.0 and 1.5 mM tetramer, (b) IHP ranging between 20 and 25 mM, and (c) IHP plus HbO2 at the
same concentrations employed in a and b. We also
carried out experiments at 0.2 mM heme concentration
(i.e. the concentration at which kinetic experiments were
performed; see below), but no appreciable difference was noticed,
indicating that the dimer-tetramer equilibrium does not affect these
results to a detectable extent. From titration curves, composed of more
than 150 experimental points and elaborated by our own programs in
order to express constant pH increments, the proton buffering
capacities (
µmol/
pH) were obtained. Buffering capacities of
IHP-bound HbO2 were computed by subtracting the contribution of IHP from the overall buffering capacities measured on
IHP plus HbO2 solution. The integration of this
differential buffering capacity gave the corrected titration curve of
IHP-bound HbO2, the position of which relative to IHP-free
HbO2 should be independently determined. Therefore, proton
uptake for the formation of the IHP-HbO2 (i.e.
Z) was obtained at several fixed pH values by measuring
the moles of HCl per mole of HbO2 needed to recover the
starting pH value after the addition to oxyHb of a saturating amount of
concentrated IHP (IHP/HbO2 molar ratio 20:1 with
[HbO2] = 1.5 mM tetramer) and the correction
for IHP dilution effects (obtained by IHP blank titration). These
values allowed us to establish the relative position of the titration
curves and thus to obtain experimental
Z values over the
whole pH range investigated (i.e. between 6.0 and 8.0).
Outside this pH range, the reproducibility of data decreased
dramatically, and thus the errors were too large to allow any
meaningful analysis of experimental curves. The value of
Z as a function of pH (see Fig. 1A) is related
to the pH dependence of the IHP binding equilibrium constant
K according to the following equation (14).
|
(Eq. 1)
|
Upon integration, this relation becomes the following
equation.
|
(Eq. 2)
|
The value of K at a given pH value (in our case,
pH = 7.1) was determined by subsequent additions of subsaturating
amounts of IHP to HbO2 and measuring after each addition
the moles of HCl needed to maintain a constant pH value. The knowledge
of the moles of HCl, of
Z at that pH, of the moles of
HbO2, and of the moles of IHP added allows one to determine
the moles of free IHP after each addition and the saturation degree of
the IHP-HbO2 complex (
). If one
assumes a single binding site for IHP to the tetrameric
HbO2 (under these experimental conditions; see Ref. 12), it
is then possible to fit values of
as a
function of x moles of free IHP (see Fig. 1B),
according to the following equation.
|
(Eq. 3)
|
where x is given by the equation,
|
(Eq. 4)
|
where CIHP is the IHP concentration of
the stock solution employed and VIHP and
Vtot are the volume added of IHP stock solution and the total volume of the sample solution, respectively. The extent
of IHP binding
is given by the following
equation.
|
(Eq. 5)
|
Thus, using Equation 2, the value of K at pH 7.1 (by
Equation 3), and
Z dependence on pH, we were able to
calculate K over the pH range between 6.0 and 8.0 (see Fig.
2).
Kinetics of O2 dissociation in fully liganded Hb was
undertaken employing a Hi-Tech SF-51 stopped-flow apparatus with a 2-cm path length observation cell that was interfaced with a desktop computer for fast data acquisition. Oxygen dissociation was followed by
mixing HbO2 (0.2 mM heme after mixing) with a
CO-saturated buffer containing sodium dithionite and following the
conversion of HbO2 to HbCO at
= 563 nm (15). No CO
concentration dependence was observed for these kinetics, down to a
concentration of 50 µM, a value 10 times lower than that
employed for all observations reported in this study (i.e.
0.5 mM after mixing). The amount of free IHP was
calculated, implying that the IHP-dependent effect on the
O2 dissociation rate constant is linearly dependent on the
percentage of IHP-HbO2 complex with respect to the total
concentration of tetrameric HbO2.
Calorimetric measurements were performed using a high-precision twin
titration isothermal microcalorimeter (16).
The Hb solution was kept inside the sample cell (total cell volume, 184 µl), and the injection syringe was filled with the concentrated IHP
solution. In order to reduce the heat of dilution, small volumes of IHP
solution (i.e. 2 µl) were added each time, and corrections
were made for the heat effects due to stirring and dilution (16).
Calibration experiments were carried out, employing HCl/NaOH titrations
and electrical calibrations (16).
Calorimetric IHP titration experiments of human HbO2 were
carried over the pH 6.0-8.0 range, employing an IHP concentration range that was enough to fully saturate the HbO2, and this
occurrence was determined when no heat was produced upon further
addition of IHP. The data analysis is based on a titration in which IHP concentration is increased at each step i from
xi
1 to xi, and
the quantity of heat qi
1 is
associated with the binding of IHP to HbO2 in this step.
The value of qi
1 is then given by
the equation,
|
(Eq. 6)
|
where mT is the moles of HbO2
employed in each calorimetric experiment.
The excess enthalpies (
00)i depend on the ligand
concentration xi according to the following
equation.
|
(Eq. 7)
|
The latter expression is a van't Hoff formulation in terms of
the binding polynomial P (17).
The heat qi
1, i is the
experimentally measurable quantity in isothermal titration calorimetry. If one assumes only one site for the interaction of IHP with
HbO2, the van't Hoff expression reduces to the following
equation.
|
(Eq. 8)
|
The value of the observed enthalpy change
Hobs, as calculated from the van't Hoff
expression, can be dissected into two main contributions, one related
to the IHP binding phenomenon itself and the other one ascribable to
the ligand-linked proton equilibria in the buffer. Therefore, because
there is a linkage between IHP binding to HbO2 and proton
release or uptake, the observed
Hobs is represented by
the equation,
|
(Eq. 9)
|
where
Hbc is the buffer-corrected enthalpy change
for IHP interaction with HbO2, which still contains the
contribution arising from the ionization enthalpy of HbO2
(18). The second term in Equation 9 refers to the apparent enthalpy
change obtained when 
moles of protons are released or taken up
to a buffer with a
Hion ionization enthalpy
change. The value of
Hbc was determined at
every pH investigated, carrying out the same calorimetric experiment in
buffers with different ionization enthalpy, such as MES, HEPES, PIPES,
Bis-Tris, and MOPS, and extrapolating to
Hion = 0 (see Equation 9 and Ref. 19).
All experiments were performed either in distilled H2O in
the presence of 0.1 M NaCl (potentiometric experiments) or
in 0.1 M MES (between pH 5.5 and 7.0) or HEPES (between pH
6.5 and 8.0) in the presence of 0.1 M NaCl (calorimetric
and kinetic experiments).
 |
RESULTS AND DISCUSSION |
Fig. 1A shows that the
total proton uptake of human HbO2 at 25 °C in the
presence of 30 mM IHP, a concentration sufficiently high to
guarantee the full saturation of the higher affinity site for IHP in
the liganded hemoglobin (11, 12), is pH-dependent, approaching 0 at pH
8.0, attaining a maximum value of
Z
2.8 at pH
7, and then decreasing upon pH
lowering. It is important to note that over the same pH range, the
buffering capacity (and thus the amount of protons exchanged with bulk
solvent) of a 30 mM solution of IHP alone was much less
than that observed for a solution of 1.5 mM tetrameric
HbO2 alone, clearly indicating that the phenomenon reported
in Fig. 1A is mostly related to the proton exchange
involving the Hb molecule and not the IHP molecule. Because
Z is the derivative of the proton-linked effect on the IHP binding constant to HbO2 (see Equation 1), a
quantitative analysis of
Z data as a function of pH (Fig.
1A) allows the determination of the linkage between IHP
binding and shifts of pKa values for groups affected
by IHP interaction with HbO2. The analysis of these data
requires the involvement of (at least) three classes of residues,
according to the following relationship.
|
(Eq. 10)
|
where x = 10
pH and
Ki = 10
pKi (i = 1-3) are
the proton binding association constants of the three groups, and the
superscript b and f refer to IHP-bound and
IHP-free HbO2, respectively. Pb and
Pf are the binding polynomials for proton binding to
IHP-bound and IHP-free HbO2, respectively.
|
(Eq. 11)
|
and
|
(Eq. 12)
|
It is important to note that Equations 10-12 imply that the three
groups are protonating in a concerted way; that is, the protonation of
the first group alters the protonation of the second group, and the
protonation of both the first and the second group affects the
protonation of the third group. In other words, groups 2 and 3, which
would not protonate in the range investigated, change their proton
affinity upon protonation of group 1. Therefore, by virtue of the
cooperative behavior, the values of Ki may be indeed
treated as intrinsic binding constants, and they can be immediately
referred to the pKa values of the various residues
involved. The pKa values of groups involved in the
IHP binding to HbO2 resulting from the fit of data in Fig. 1A according to Equation 10 are reported in Table
I, and they correspond to the continuous
line in Fig. 1A. It is important to note that in Table I the
pK3 for IHP-free HbO2 is reported only as being <4.5, because any value below 4.5 gives an equally good
fit of data, and we can consider its value as partially
undetermined.

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Fig. 1.
A, observed pH dependence at 25 °C of
proton uptake or release ( Z) upon binding of saturated
amounts of IHP to HbO2. The error bars refer to
the distribution of errors based on five different measurements of the
same sample. The data presented are limited to the pH range between 6.0 and 8.0 because outside this range, data become very unreliable. The
continuous line corresponds to the behavior expected for
Equation 10, employing the parameters reported in Table I. Dashed
line corresponds to the fit of data employing only two protonating
groups. For further details, see text. B, saturation
function ( ) of HbO2 as a function
of IHP addition at pH 7.1 and 25 °C. The continuous line
was obtained by nonlinear least-squares fitting of experimental data
according to Equation 3. The fitted limiting values for
= 1.0 (i.e. under saturating
amounts of IHP) have been employed to calculate Z at the
given pH. For further details, see text.
|
|
Fig. 1B displays the fitting of pH-stat data on the
equilibrium titration of human HbO2 with IHP at pH 7.1 according to Equation 3, which allows one to calculate the affinity of
IHP for HbO2 at this pH. Combination of the information
obtained from the experiments reported in the two panels of Fig. 1,
namely (a)
Z as a function of pH (Fig.
1A), and (b) the equilibrium IHP binding constant at a given pH value (Fig. 1B), allows one to calculate,
according to Equation 2 (14), the logK for IHP binding to
human HbO2 over the pH range covered by the proton
titration reported in Fig. 1A. In Fig. 2, the pH dependence
of the equilibrium IHP binding constant to human HbO2 is
reported at 25 °C. The continuous line reported in Fig.
2 was obtained employing the following
equation.
|
(Eq. 13)
|
where Kobs is the observed IHP equilibrium
binding constant, K0 is the IHP equilibrium
binding constant to unprotonated HbO2, and
Pb and Pf are the proton binding
polynomials to IHP-bound and IHP-free human HbO2,
respectively, (see Equations 11 and 12), employing the values of
Ki reported in Table I. Therefore, the
interrelationship between IHP and proton linkage can be represented by
the following Scheme.
Scheme I and the pKa values reported in Table I
deserve some further comment. As a matter of fact, the behavior observed in Table I underlies a cooperative proton-linked process, such
that protonation of one residue facilitates the protonation of another
residue. This concerted process may envisage the occurrence of a
pH-dependent conformational transition in liganded human Hb, as also suggested by previous observations (15). Furthermore, Scheme I indicates that IHP and protons act synergistically to facilitate the conformational transition, raising the
pKa of interacting groups upon binding of the
negatively charged IHP. Obviously, with our experimental approach, we
cannot absolutely rule out a contribution arising, in addition, from a
change in the protonation state between Hb-free and Hb-bound IHP, even
though the small amount of proton ex- changed by IHP alone (see
above) indicates that this contribution is not relevant. This
conclusion is further supported by a previous observation on deoxy Hb
and on HbCO by 31P NMR, where a change in the protonation
state of IHP upon binding Hb indeed was detected, but it turned out to
be pH-independent between pH 5.2 and 8.5 (20). Therefore, the observed
pH dependence for IHP binding to HbO2 (see Fig. 2) can be
almost completely attributed to a pH-dependent difference
in protons bound by IHP-free and IHP-bound oxyHb.

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Fig. 2.
pH dependence of IHP binding constant to
HbO2 in 0.1 M NaCl, at 25 °C, according to
Equation 2 and employing data reported in Fig. 1. The
line represents the least-squares fitting of data, employing
Equation 13, with pKa values reported in Table I and
K0 = 5.13 × 102
M 1. For further details, see text.
|
|
This proton-linked behavior is calculated on the basis of the
proton titration carried out on IHP-free and IHP-bound human HbO2, but a confirmation of its validity may come from an
independent measurement of IHP binding to fully liganded
HbO2. This can be carried out by investigating the effect
of IHP on the displacement kinetics of oxygen by CO. Thus, in this
experimental approach, the rate of CO binding is rendered much faster
than the O2 dissociation process, and the observation
concerns a fully liganded protein, allowing a direct determination of
IHP binding to HbO2. In Fig. 3, the values of rate
constants for O2 dissociation from fully liganded Hb are
reported as a function of free IHP concentration at different pH
values. It is important to note that the continuous lines in Fig.
3 are not fit to experimental points;
instead, they simply show the correlation between the predicted pH
dependence of K (see Fig. 2) and the observed pH dependence
of the IHP effect on the O2 dissociation rate constant from
fully liganded Hb. Therefore, they are constrained to the expected IHP
dependence on the basis of the IHP binding equilibrium constant at the
same pH according to the parameters reported in Table I, employed to
fit the data reported in Fig. 1A, and used to obtain the
continuous line in Fig. 2. The agreement is quite impressive and allows
a very strong degree of confidence in the correctness of the prediction
based on data in Fig. 1A and on Equation 2, and thus in the
accuracy of parameters in Table I, as well as in the pH dependence
described in Fig. 2, to quantitatively describe the linkage between
proton and IHP binding to human HbO2.

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Fig. 3.
Dependence of O2 dissociation
rate constant from HbO2 on IHP concentration at different
pH values, in 0.1 M NaCl, at 25 °C, and at pH 6.1 (in
0.1 MES) (×), pH 6.4 (in 0.1 M MES)
(+), pH 6.7 (in 0.1 MES) (*), 7.1 (in 0.1 M HEPES) ( ), and pH 7.4 (in 0.1 M HEPES)
( ). The lines correspond to a simple binding
process, as described by the equation: kobs = k0/P + kIHP
· K · [IHP]f/P, where
kobs is the observed O2 dissociation
rate constant, k0 is the dissociation rate
constant observed in the absence of IHP, K is the IHP
binding constant to human HbO2, as derived from the curve
reported in Fig. 2, kIHP is the O2
dissociation rate constant observed in 30 mM IHP (and
corresponding to IHP-bound HbO2), and P (= 1 + K · [IHP]f) is the binding polynomial for the
IHP interaction with HbO2. The line was obtained
imposing at every pH value (a) k0,
obtained experimentally in the absence of IHP, and (b)
K, as obtained at the given pH by the fitting curve reported
in Fig. 2. kIHP was the only floating parameter,
and values obtained at every investigated pH are shown in Fig. 4 for
IHP-bound HbO2. For further details, see text.
|
|
Parameters in Table I clearly indicate that the pH-dependence of
IHP binding constant depends on the pKa shift of three classes of residues that increase their pKa
values by
0.96, 0.92, and >3.7, respectively, upon interaction with negative charges of IHP. It is important to note that in free HbO2 the pKa values of at least two of
these residues turn out to be low enough to rule out the relevant role
in the "alkaline" Bohr effect, whereas a third residue displays a
pKa of 6.72 (see Table I) in the IHP-free
HbO2, which makes it a good candidate for a contribution to
the alkaline Bohr effect (21-23). On the other hand, such
pKa values for IHP-free HbO2 are within
the pH range of a conformational transition, which has been detected in
human HbO2 in the absence of anions (15) and which is
characterized by an enhancement of the O2 dissociation rate
constant in the fully liganded form. A similar behavior was observed in
the presence of 0.1 M Cl
(Fig.
4), and it can be accounted for by
employing the three pKa values reported in Table I
for IHP-free HbO2, as from the continuous line reported in
Fig. 4. The same consideration can be applied to the pH dependence of
the O2 dissociation rate constant for fully liganded Hb in
the presence of saturating amounts of IHP (i.e. 30 mM), which also is fully described employing the pKa values reported in Table I for IHP-bound human
HbO2 (see Fig. 4).

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Fig. 4.
pH dependence of the O2
dissociation rate constant from HbO2, in 0.1 M
NaCl, at 25 °C, from IHP-free (×) and IHP-bound human
HbO2 ( ). These data correspond to the values of
k0 and kIHP in the
previous figure, at the corresponding pH values. Buffers employed were
0.1 M MES for pH 7.0 and HEPES for pH 7.0. The lines correspond to the following equation:
kobs = k1/P + k2 · K1[H+]/P + k3 · K1 · K2[H+]2 + k4 · K1 · K2 · K3[H+]3, where
kobs is the observed O2 dissociation
rate constant from HbO2, k1,
k2, k3, and
k4 are the O2 dissociation rate
constants from unprotonated, singly protonated, doubly protonated, and
triply protonated HbO2, respectively (and were the only
free-floating parameters), P is the binding polynomial for
proton binding to either IHP-free (see Equation 12) or IHP-bound (see
Equation 11) HbO2. Values of K1,
K2, and K3 were those
reported in Table I for the IHP-free and IHP-bound HbO2.
For IHP-free HbO2, the continuous curve was obtained using
k1 = 15.3 s 1,
k2 = 37.3 s 1,
k3 = 72.3 s 1, and
k4 = 130 s 1. For IHP-bound
HbO2, the continuous curve was obtained using
k1 = 15.3 s 1,
k2 = 50 s 1,
k3 = 90 s 1, and
k4 = 130 s 1. However, it must be
noticed that in the case of IHP-bound HbO2, the values of
k2 and k3 are very poorly
defined, because the curve is scarcely affected by changes of their
values. For further details, see text.
|
|
Altogether, these data strengthen our confidence in the
possibility of giving a quantitative description of the thermodynamics of IHP interaction with human HbO2. Therefore, we can claim
that (a) the protonation of three residues, the pKa
of which values range in IHP-free HbO2 between
4.0 and
6.7 (see Table I), brings about a conformational transition in fully
liganded human Hb, (b) this event is closely related to the
pH-dependent enhancement of the IHP equilibrium binding
constant to HbO2 (Fig. 2), and (c) IHP binding
is accompanied by a more or less marked raising of
pKa values of these three classes of residues.
The identification of the three residues involved in the
proton-linked IHP binding to HbO2 is not easy, but previous
observations indicated that some potentially important residues display
low pKa values in oxyHb in the absence of organic
phosphates (24). In particular, a fairly low pKa
value (i.e. pKa < 4.5) has been reported by several
authors for His143
in HbO2 (25), a residue
that has been already proposed to be involved in the binding of organic
phosphate (6, 10). A second residue might be Val1
, which
has been proposed to display a pKa
6.8 in
IHP-free HbO2 (25) and which might be tentatively
recognized in the residue characterized by a pKa = 6.72 (see Table I). The third residue (characterized by a
pKa = 5.96 in IHP-free HbO2; see Table
I) is very difficult to identify, even with some uncertainty, and we
cannot rule out at this stage that the effect attributed to this
IHP-linked group is instead attributable to a widespread small effect
on several residues, such as His72
and
His77
, which have been reported to have in HbO2
pKa values below 6.5 (25). However, it must
be pointed out that a fairly low pKa < 6.5 has been
also proposed for His2
(24, 25), another residue in the
-dyad axis cavity where organic phosphates bind (6, 10). The
possibility of a role by His2
in the interaction of IHP
with HbO2 is not in contradiction with the observation on a
mutant, namely Hb Deer Lodge (where His2
is substituted by Arg; see
Ref. 26), in which the IHP effect on oxygenation appears unmodified.
Thus, (a) Arg may substitute reasonably well for His in the
interaction, such that the effect of the substitution is substantially
reduced, and (b) in the oxygenation, an effect is observed
only if there is a difference in the IHP binding mode between deoxy-
and oxyHb, and this seems to be not true for His2
(24,
25). The role of Lys82
has not been taken into account
in our analysis of the pH dependence simply because its
pKa is much too high to come into play over the pH
range investigated (25, 27), but its contribution to the free energy of
IHP binding is probably a major one in determining the affinity for
pH > 8.0.
The linkage relationship between proton and IHP interaction with human
HbO2 can be described in quantitative energetic terms by
calorimetric measurements of the heat that accompanies binding of IHP
at different pH values. In this way, information concerning
H of the
interaction allowed us to attempt a correlation between (a)
protonation of residues in IHP-free and IHP-bound HbO2,
(b) the free energy involved in the interaction, and
(c) the entropic contribution to the binding process. Fig.
5 shows such relationships in the pH
range between 6 and 8, from which it was concluded that (a)
at pH < 7.0, the IHP binding is essentially enthalpy-driven (
H
being strongly exothermic and pH-independent, with a value of 
59
kJ/mol), whereas the pH dependence of
G is completely attributable
to the pH dependence of
S, which is always positive for
values of pH
7.0; (b) at pH > 7.0 there is a
progressive decrease of
S, which becomes negative at
pH > 7.4, accompanied by a decrease of the exothermicity of the
process, which becomes endothermic at pH > 7.5. Therefore, at
pH > 7.0, the entropy role in determining the affinity of IHP for
HbO2 becomes progressively predominant as pH increases, and
a proton-linked enthalpy-entropy compensation comes into play in
regulating the pH dependence of the free energy for IHP binding.
Therefore, it appears as if two different interaction modes are
operative in modulating the IHP binding, one predominating at pH < 7.0 and the other predominating at pH > 7.0. We must stress at
this point that a previous calorimetric investigation of the
interaction of IHP with HbCO at few pH values gave results fully
compatible with ours, at least at the corresponding pH values (28).

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Fig. 5.
Values of G (+), H ( ) and
T S (*) for IHP binding to human HbO2 in 0.1 M NaCl, at 25 °C. At every pH, values of G were
obtained from K, as derived in Fig. 2, values of H were
obtained from calorimetric measurements (see Equation 9 under
"Experimental Procedures"), and values of T S were derived
according to the relationship T S = H G.
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Altogether, this behavior may be tentatively correlated with the
pKa shifts reported above (see Table I). Thus, at
very alkaline pH (i.e.
8.0), IHP interaction is not
accompanied by any proton release or uptake, and it appears to be an
endothermic process, displaying a negative
S. As the pH
is decreased toward 7.0, the three IHP-linked protonating groups take
up protons when IHP interacts with HbO2, increasing the
Z (see Fig. 1B). The proton uptake during IHP
binding also brings about a progressive decrease of the endothermicity,
with a
H < 0 at pH < 7.5, mirrored by a
parallel increase of
S, which becomes positive at pH < 7.4. (see Fig. 5). As the pH is lowered below 7.0, the three
IHP-linked protonating groups begin to take up protons in IHP-free
HbO2 as well, corresponding to a decrease of
Z (Fig. 1B). Such a process seems to affect
the
S of IHP interaction, decreasing its positive value
and thus increasing the
G of binding, whereas
H appears not to
depend on the protonation of these groups in IHP-free HbO2 (Fig. 5).
Therefore, it seems that the progressively increasing exothermicity of
IHP interaction upon pH lowering indeed may be related to the heat
released by the groups that take up protons when IHP binds (29).
However, the pH-independent value of
H at pH < 7.0, over a
range in which the groups are already protonated in IHP-free
HbO2 and the extent of IHP-linked proton uptake
(i.e.
Z) decreases, seems to suggest that
additional factors might come into play to determine the observed
exothermicity of IHP binding at low pH, such as the electrostatic
interaction between the positive charges of HbO2 and the
negative charges of IHP.