Thermodynamics of Inositol Hexakisphosphate Interaction with Human Oxyhemoglobin*

Irene MessanaDagger , Mauro Angeletti§, Massimo Castagnola, Giampiero De Sanctis§, Enrico Di StasioDagger , Bruno GiardinaDagger , Stefania Pucciarelli§, and Massimo Colettaparallel **

From the Dagger  Consiglio Nazionale delle Ricerche Center for Receptor Chemistry and Institute of Chemistry and Clinical Chemistry, Catholic University of Sacred Heart, Largo F. Vito 1, 00168 Rome, Italy, the § Department of Molecular, Cellular and Animal Biology, University of Camerino, V. F. Camerini 2, 62032 Camerino (MC), Italy, the  Institute of Biological Chemistry, University of Cagliari, 09126 Cagliari, Italy, and the parallel  Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, 00133 Rome, Italy

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

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 Val1beta and His143beta , which are located in the beta 1beta 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
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Abstract
Introduction
Procedures
Results & Discussion
References

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 beta -chains, mainly involving three residues from either one of beta -subunits (i.e. HisNA2(beta 2), LysEF6(beta 82), and HisH21(beta 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
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Abstract
Introduction
Procedures
Results & Discussion
References

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 (delta  µmol/delta 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. Delta 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 Delta 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 Delta 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).
<UP>dlog</UP>K/<UP>dpH</UP>=<UP>−</UP>&Dgr;Z (Eq. 1)
Upon integration, this relation becomes the following equation.
<UP>log</UP> K(<UP>pH</UP><SUB>1</SUB>)−<UP>log</UP> K(<UP>pH</UP><SUB>2</SUB>)=<LIM><OP>∫</OP><LL><UP>pH</UP><SUB><UP>1</UP></SUB></LL><UL><UP>pH</UP><SUB><UP>2</UP></SUB></UL></LIM>&Dgr;Z <UP>dpH</UP> (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 Delta 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 (<OVL><IT>Y</IT></OVL>). 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 <OVL><IT>Y</IT></OVL> as a function of x moles of free IHP (see Fig. 1B), according to the following equation.
<A><AC>Y</AC><AC>&cjs1171;</AC></A>=Kx/(1+Kx) (Eq. 3)
where x is given by the equation,
x=(C<SUB><UP>IHP</UP></SUB> · V<SUB><UP>IHP</UP></SUB>−(&mgr;<UP>mol of HCl/</UP>&Dgr;Z))/V<SUB><UP>tot</UP></SUB> (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 <A><AC>Y</AC><AC>&cjs1171;</AC></A> is given by the following equation.
<A><AC>Y</AC><AC>&cjs1171;</AC></A>= <UP>&mgr;mol of HCl/</UP>&Dgr;Z/<UP>&mgr;mol of HbO<SUB>2</SUB></UP> (Eq. 5)
Thus, using Equation 2, the value of K at pH 7.1 (by Equation 3), and Delta 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 lambda  = 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,
q<SUB>i<UP>−</UP>1,i</SUB>=m<SUB><UP>T</UP></SUB>[(<A><AC>H</AC><AC>&cjs1171;</AC></A>−<A><AC>H</AC><AC>&cjs1171;</AC></A><SUB>00</SUB>)<SUB>i</SUB>−(<A><AC>H</AC><AC>&cjs1171;</AC></A>−<A><AC>H</AC><AC>&cjs1171;</AC></A><SUB>00</SUB>)<SUB>i<UP>−</UP>1</SUB>] (Eq. 6)
where mT is the moles of HbO2 employed in each calorimetric experiment.

The excess enthalpies (<A><AC>H</AC><AC>&cjs1171;</AC></A> - <A><AC>H</AC><AC>&cjs1171;</AC></A>00)i depend on the ligand concentration xi according to the following equation.
(<A><AC>H</AC><AC>&cjs1171;</AC></A>−<A><AC>H</AC><AC>&cjs1171;</AC></A><SUB>00</SUB>)<SUB>i</SUB>=<UP>−</UP>R(∂<UP>ln</UP>P/∂1/T)<SUB>x</SUB> (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.
(<A><AC>H</AC><AC>&cjs1171;</AC></A>−<A><AC>H</AC><AC>&cjs1171;</AC></A><SUB>00</SUB>)=<UP>−</UP>R(&dgr;<UP>ln</UP>P/&dgr;1/T)<SUB>x</SUB>=&Dgr;H · Kx/(1+Kx) (Eq. 8)

The value of the observed enthalpy change Delta 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 Delta Hobs is represented by the equation,
&Dgr;H<SUB><UP>obs</UP></SUB>=&Dgr;H<SUB><UP>bc</UP></SUB>+&Dgr;&ngr;H<SUP><UP>+</UP></SUP> · &Dgr;H<SUB><UP>ion</UP></SUB> (Eq. 9)
where Delta 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 Delta nu moles of protons are released or taken up to a buffer with a Delta Hion ionization enthalpy change. The value of Delta 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 Delta 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
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Abstract
Introduction
Procedures
Results & Discussion
References

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 Delta Z congruent  2.8 at pH approx  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 Delta Z is the derivative of the proton-linked effect on the IHP binding constant to HbO2 (see Equation 1), a quantitative analysis of Delta 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.
&Dgr;Z=((<SUP>b</SUP>K<SUB>1</SUB>x+2<SUP>b</SUP>K<SUB>1</SUB><SUP>b</SUP>K<SUB>2</SUB>x<SUP>2</SUP>+3<SUP>b</SUP>K<SUB>1</SUB><SUP>b</SUP>K<SUB>2</SUB><SUP>b</SUP>K<SUB>3</SUB>x<SUP>3</SUP>)/P<SUB>b</SUB>)−((<SUP>f</SUP>K<SUB>1</SUB>x+2<SUP>f</SUP>K<SUB>1</SUB><SUP>f</SUP>K<SUB>2</SUB>x<SUP>2</SUP>+3<SUP>f</SUP>K<SUB>1</SUB><SUP>f</SUP>K<SUB>2</SUB><SUP>f</SUP>K<SUB>3</SUB>x<SUP>3</SUP>)/P<SUB>f</SUB>) (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.
P<SUB>b</SUB>=(1+<SUP>b</SUP>K<SUB>1</SUB>[<UP>H<SUP>+</SUP></UP>]+<SUP>b</SUP>K<SUB>1</SUB> · <SUP>b</SUP>K<SUB>2</SUB>[<UP>H<SUP>+</SUP></UP>]<SUP>2</SUP>+<SUP>b</SUP>K<SUB>1</SUB> · <SUP>b</SUP>K<SUB>2</SUB> · <SUP>b</SUP>K<SUB>3</SUB>[<UP>H<SUP>+</SUP></UP>]<SUP>3</SUP>) (Eq. 11)
and
P<SUB>f</SUB>=(1+<SUP>f</SUP>K<SUB>1</SUB>[<UP>H<SUP>+</SUP></UP>]+<SUP>f</SUP>K<SUB>1</SUB> · <SUP>f</SUP>K<SUB>2</SUB>[<UP>H<SUP>+</SUP></UP>]<SUP>2</SUP>+<SUP>f</SUP>K<SUB>1</SUB> · <SUP>f</SUP>K<SUB>2</SUB> · <SUP>f</SUP>K<SUB>3</SUB>[<UP>H<SUP>+</SUP></UP>]<SUP>3</SUP>) (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 (Delta 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 (<OVL><IT>Y</IT></OVL>) 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 <OVL><IT>Y</IT></OVL> = 1.0 (i.e. under saturating amounts of IHP) have been employed to calculate Delta Z at the given pH. For further details, see text.

                              
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Table I
pKa values of IHP-linked protonating groups in the IHP-free and IHP-bound HbO2 at 25 °C in 0.1 M NaCl

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) Delta 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.
K<SUB><UP>obs</UP></SUB>=K<SUB>0</SUB> · P<SUB>b</SUB>/P<SUB>f</SUB> (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.
<AR><R><C><UP>     P</UP>+<UP>H</UP><SUP>+</SUP> <LIM><OP><ARROW>⇌</ARROW></OP><UL><UP>p<SUP>f</SUP>K<SUB>1</SUB></UP></UL></LIM><UP> PH</UP>+<UP>H</UP><SUP>+</SUP> <LIM><OP><ARROW>⇌</ARROW></OP><UL><UP>p<SUP>f</SUP>K<SUB>2</SUB></UP></UL></LIM><UP> PH<SUB>2</SUB></UP>+<UP>H</UP><SUP>+</SUP> <LIM><OP><ARROW>⇌</ARROW></OP><UL><UP>p<SUP>f</SUP>K<SUB>3</SUB></UP></UL></LIM> <UP>PH</UP><SUB><UP>3</UP></SUB></C></R><R><C>            +                   +                     +                      +</C></R><R><C><UP>     IHP       IHP        IHP         IHP</UP></C></R><R><C>           ⥮<SUP>0</SUP>K<SUB><UP>IHP</UP></SUB>        ⥮<SUP>1</SUP>K<SUB><UP>IHP</UP></SUB>         ⥮<SUP>2</SUP>K<SUB><UP>IHP</UP></SUB>             ⥮<SUP>3</SUP>K<SUB><UP>IHP</UP></SUB></C></R><R><C></C></R><R><C><UP>PIHP</UP>+<UP>H </UP><SUP>+ </SUP><LIM><OP><ARROW>⇌</ARROW></OP><UL>   </UL></LIM><UP> HPIHP</UP>+<UP>H</UP><SUP>+</SUP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>   </UL></LIM> <UP>H</UP><SUB>2</SUB><UP>PIHP</UP>+<UP>H </UP><SUP>+</SUP><LIM><OP><ARROW>⇌</ARROW></OP><UL>   </UL></LIM><UP> H</UP><SUB>3</SUB><UP>PIHP</UP></C></R><R><C><UP>        p<SUP>b</SUP>K<SUB>1</SUB>         p<SUP>b</SUP>K<SUB>2</SUB>          p<SUP>b</SUP>K</UP><SUB><UP>3</UP></SUB></C></R></AR>
<UP><SC>Scheme I</SC></UP>
<SUP>i</SUP>K<SUB><UP>IHP</UP></SUB>=<SUP>0</SUP>K<SUB><UP>IHP</UP></SUB><FENCE><LIM><OP>∏</OP><LL>n<UP>=</UP>1</LL><UL>n<UP>=</UP>i</UL></LIM><SUP> b</SUP>K<SUB>n</SUB>/<LIM><OP>∏</OP><LL>n<UP>=</UP>1</LL><UL>n<UP>=</UP>i</UL></LIM><SUP> f</SUP>K<SUB>n</SUB></FENCE>.
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) (open circle ), and pH 7.4 (in 0.1 M HEPES) (oplus ). 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 congruent 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 (open circle ). 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 approx 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 His143beta 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 Val1beta , which has been proposed to display a pKa approx  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 His72alpha and His77beta , 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 His2beta (24, 25), another residue in the beta -dyad axis cavity where organic phosphates bind (6, 10). The possibility of a role by His2beta in the interaction of IHP with HbO2 is not in contradiction with the observation on a mutant, namely Hb Deer Lodge (where His2beta 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 His2beta (24, 25). The role of Lys82beta 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 Delta 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 (Delta H being strongly exothermic and pH-independent, with a value of congruent -59 kJ/mol), whereas the pH dependence of Delta G is completely attributable to the pH dependence of Delta S, which is always positive for values of pH <=  7.0; (b) at pH > 7.0 there is a progressive decrease of Delta 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 Delta G (+), Delta H (open circle ) and TDelta S (*) for IHP binding to human HbO2 in 0.1 M NaCl, at 25 °C. At every pH, values of Delta G were obtained from K, as derived in Fig. 2, values of Delta H were obtained from calorimetric measurements (see Equation 9 under "Experimental Procedures"), and values of TDelta S were derived according to the relationship TDelta S = Delta H - Delta G.

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 Delta 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 Delta Z (see Fig. 1B). The proton uptake during IHP binding also brings about a progressive decrease of the endothermicity, with a Delta H < 0 at pH < 7.5, mirrored by a parallel increase of Delta 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 Delta Z (Fig. 1B). Such a process seems to affect the Delta S of IHP interaction, decreasing its positive value and thus increasing the Delta G of binding, whereas Delta 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 Delta 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. Delta 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.

    ACKNOWLEDGEMENT

We thank Professor Ascenzi for several stimulating discussions.

    FOOTNOTES

* This study was supported by the Italian Ministero dell'Universitá e della Ricerca Scientifica e Tecnologica and the Italian National Research Council (Consiglio Nazionale delle Ricerche).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 39-6-72596365; Fax: 39-6-72596353; E-mail: massimo.coletta{at}uniroma2.it.

1 The abbreviations used are: Hb, hemoglobin; IHP, inositol hexakisphosphate; 2,3-DPG, 2,3-diphosphoglycerate; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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