Calorimetric Studies of Carbon Monoxide and Inositol Hexaphosphate Binding to Hemoglobin A*

Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated. For IHP binding to deoxyhemoglobin the buffer-cor-rected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may arise largely from the protonation of histidine and NHz-terminal groups in the binding site (Arnone, A., and Perutz, M. F. (1974) Nature 249, 34-36). The decreased magnitude of AHIHP observed at low pH parallels the decreased proton uptake at low pH. In 0.1 M chloride (pH 7.4) the reaction has a standard free energy change R. E., Benesch, R. (1976) J. Biol. Chem. 251, 7720-7721) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phos- phates to hemoglobin may well extend


Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated.
For IHP binding to deoxyhemoglobin the buffer-corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may arise largely from the protonation of histidine and NHz-terminal groups in the binding site Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.
Thermodynamics of hemoglobin-ligand-binding reactions can be analyzed profitably in terms of the allosteric model of Monod et al. (1965). Central to this model is the notion that the energetics of interaction of any substance wit.h hemoglobin differ according to the hemoglobin conformational state. Such differential affinity of substances like buffer ions for hemoglobin in the oxy and deoxy states provides the linkage by which solution components affect the more important hemoglobinligand-binding reactions such as oxygen and organic phosphate binding. The general effects of buffer ions, pH, etc., on equilibrium constants of these reactions have been studied for over a decade (Antonini and Brunori, 1971;Benesch and Benesch, 1974). . of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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In order to explore the importance of the enthalpy and entropy contributions to the free energy change of ligandbinding reactions, we have directed our attention to the enthalpic components of hemoglobin-ligand reactions. Calorimetric studies of how solution conditions affect heats of hemoglobin-ligand reactions have been particularly limited. Rudolph andGill (1974) andNelson et al. (1974) examined pH effects on heats of CO and IHP binding, respectively, to hemoglobin A. Atha and Ackers (1974) evaluated the differential heat of chloride interaction with deoxy and 0 2 hemoglobin. Gaud et al. (1975) have attempted to assess buffer effects on the heat of CO binding by HbM Iwate. The experience of these studies shows that a variety of solution conditions such as pH, buffer species, and ionic strength influence the value of the enthalpy change for various ligands. Nonetheless, a comprehensive and self-consistent set of data has been lacking.
We have therefore examined heats of CO and IHP binding to hemoglobin A under a variety of buffer and pH conditions. From these data we are able to estimate and compare the heats of ion binding linked to hemoglobin oxygenation and organic phosphate binding. The results of this study shed interesting light on the sources of the thermodynamic driving force for hemoglobin-allosteric effector interactions. In particular, we find that the enthalpic contribution for binding of IHP to hemoglobin is directly related to proton uptake in the reaction.

MATERIALS AND METHODS
Freshly drawn hemoglobin A was prepared as described by Benesch et al. (1968). The hemoglobin was then either dialyzed against a buffer solution or unbuffered 0.1 M NaCl or, in the case of unbuffered, deionized solutions, it was dialyzed three times against distilled water and then deionized on a Bio-Rex AG 501-X8 mixed bed resin from Bio-Rad Laboratories. The pH was then adjusted with 0.1 M acid or base. The samples were deoxygenated in a tonometer with a cuvette attached and examined spectrally. Details are given by Gaud et nl. (1975). The hemoglobin concentration was adjusted to 1 to 2 mM heme. No reducing agents were used since the spectra indicated methemoglobin to he less than 2%'. Furthermore, we wished to avoid introduction of any additional ionic species.
The IHP was purchased from P-L Biochemicals as sodium inositol hexaphosphate and analyzed by Huffman Laboratories, Wheatridge, col., to ascertain water content. Solutions of 25 mM were prepared by weighing the IHP and mixing it with dialysate buffer. The pH was adjusted to that of the original buffer.
The gas-liquid microcalorimeter was described by Rudolph et al. (1972) and improvements are described by Rudolph and Gill (1974) and Gaud et al. (1975). The procedures for liquid titrations and for CO gas titrations are given by No11 et al. (1979). An example of thermal titration with IHP is shown in Fig. 1 (Berg, 1975) and -13.34 kcal/mol (Larson and Hepler, 1969), respectively.

RESULTS AND DISCUSSION
Analysis of calorimetric data for CO and IHP binding first requires recognizing that protons released or absorbed in the reactions are abstracted from or absorbed by the buffer with an associated observable heat change. Calculation of this heat effect requires a knowledge of the change in hemoglobin protonation during the reaction. Table I summarizes values obtained by Brygier et al. (1975) for proton uptake by hemoglobin for reactions involving CO and IHP binding a t various pH values. Multiplying the change in protonation by the buffer ionization heat (Christensen et al., 1976) yields the contribution AHHuII to the observed heat AH0bs due to the protonation change of the buffer. Subtraction of AHB"~~ from AHOhh yields the buffer corrected heat AHBc. When unbuffered hemoglobin solutions were examined, the apparent heat of hemoglobin ionization (Antonini et al., 1965) was used in place of the heat of proton ionization of the buffer.
We measured calorimetrically the heats of the following four reactions in 0.1 M chloride-buffered Tris and bis-Trisl and self-buffered at three pH values: Hb(aq) + 4 CO(g) + Hb(CO)r(aq), 4AH~:~(kcal/mol CO) (1) mol of CO is similar to the -17.5 kcal/mol of CO value observed previously (Rudolph and Gill, 1974) but under different solution conditions. A value of -15.5 kcal/mol of CO is estimated from oxygen-binding heats determined by Atha and Ackers (1974) at pH 7.5. This calculation involves correction for heat of CO solution (-3.0 kcal/mol of CO) and for the heat of replacement of bound Os by CO (-4.0 kcal/mol of CO).
For IHP binding to deoxyhemoglobin, the buffer corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may arise largely from the protonation of histidine and NHz-terminal groups in the binding site (Arnone and Perutz, 1974). The decreased magnitude of AHIH~ observed at low pH parallels the decreased proton uptake at low pH. A similar effect is seen for IHP binding to CO-ligated hemoglobin where observed enthalpy changes average about -8 kcal/mol of H' absorbed.
The four reactions constitute a thermodynamic cycle such that 4 AHCO + AH:$:. should equal 4 AH:%' + AHIH:.. The difference is given as Zcycle in the right column of Table I1 and is well within experimental error. Differential ion binding by ligated and unligated hemoglobin gives rise to substantial enthalpic effects. For each reaction the buffer correction term was calculated. In the absence of ligand-linked ion binding, all reactions should exhibit ideal enthalpy changes AHidea1 equal to that observed for deionized hemoglobin plus the difference between the buffer correction terms for the solution of interest and the deionized hemoglobin. The difference between the observed and ideal heat of ligand binding represents the heat AH,,, of ligand-linked ion binding. Table I11 shows this analysis of CO binding by HbA for various buffers at pH 7.4. We may interpret the uniformly endothermic quantities in the right column as being the heats of anion loss from deoxyhemoglobin which accompanies CO binding. The data show CO-linked chloride ion-binding heats to hemoglobin to be about +2.8 kcal/mol of CO. This accords well with the value of +3.0 kcal determined by Atha and Ackers (1974). In the case of 0 2 binding, about 0.4 mol of C1is released/mol of O2 bound (Van Beek et al., 1979). If a similar value obtains for CO then the actual heat of C1binding to deoxyhemoglobin is -7.5 kcal/mol of ClF.
The thermal effects of ligand-linked ion binding in phosphate and maleate buffers are substantially larger than ob-  of 4.0 and 4.6 kcal/mol of CO, respectively. In the case of phosphate the same value (4.6 kcal/mol of 0 2 ) has been obtained for 0.1 M phosphate containing no chloride by Atha and Ackers (1974). The implication of these results is that, in phosphate-containing solutions, the higher charge may simply contribute to a more exothermic interaction with the deoxyhemoglobin. This might be due to enhanced protonation of nitrogen groups in the allosteric site as has been suggested for the case of IHP binding (No11 et al., 1979).
The binding of IHP to deoxyhemoglobin must necessarily displace any ions originally bound at the allosteric site. The approach used in analyzing heats of CO-linked ion binding to hemoglobin can be used to estimate thermal effects associated with IHP-linked ion release, The results of this analysis at pH 7.4 are shown in Table IV. The heat of chloride ion binding as replaced by IHP ranges from -4.1 to -8.0 kcal/mol of IHP in 0.1 M chloride. Tris and bis-Tris give similar values of -4.1 and -4.7 kcal/mol of IHP, respectively, while in the unbuffered solution yields a value of -8.0 kcal/mol of IHP. The higher value observed for the unbuffered case would seem to imply a specific effect of the his-Tris and Tris buffers on the hemoglobin. On the other hand, since the probable error in these values is in the range of 2 kcal, the differences in the values of AH,,, for the three chloride-containing solutions would barely be significant.
The significantly large heats of AH,,, observed for maleate and phosphate solutions are ascribed to the same factors noted for CO binding, namely increased ion release due to stronger binding and/or higher evolution per ion bound. Both of these factors arise from the higher negative charges of phosphate and maleate which by requiring increased ionic interaction on binding give rise both to high affinity and more exothermic heats of interaction than is possible for a singly charged species like chloride.
It seems unlikely (Van Beek et al., 1979) for phosphate and maleate that more than two anions are displaced upon IHP binding. If one assumes that two anions are indeed bound in the allosteric site then about 10 kcal are evolved for each anion bound. Heats of such magnitude suggest concomitant protonation as indeed has been observed with the binding of IHP itself.   Table V shows the comparison of the differential heats of anion binding to hemoglobin for CO and IHP ligation to deoxyhemoglobin. We note that there is rough agreement between the energetics of ion displacement by the two processes. Such behavior would be expected since on the one hand the binding of IHP to the allosteric site will displace any anions there bound and on the other hand experimental studies of CO binding show that, at least for chloride, both high affinity chloride ions are released. Thus, both CO and IHP ligation of hemoglobin lead essentially to the same anion displacement and should give rise to similar thermal effects. The magnitude of the thermal effect of IHP binding to deoxyhemoglobin merits further consideration. In 0.1 M chloride (pH 7.4) the reaction Hb(aq) + IHP + Hb.IHP(aq) has a standard free energy change (Edalji et al., 1976) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by t.he proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.