Thermodynamic studies on oxygen binding by human red blood cells

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<ul><li><p>Comparative Biochemistry and Physiology Part A 123 (1999) 329336</p><p>Thermodynamic studies on oxygen binding by human red bloodcells</p><p>Pierre Vorger *Ser6ice de Physiologie Respiratoire, Pa6illon Beauchant, Hopital Jean Bernard, B.P. 577, F-86021 Poitiers Cedex, France</p><p>Received 27 December 1998; received in revised form 16 April 1999; accepted 25 April 1999</p><p>Abstract</p><p>Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and41C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive stepsof oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. Onthe nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88118]. The heat release appears to be nearly equal forthe four steps. At the first three steps, the DH change is counterbalanced by a nearly equivalent change of DS, resulting in a rathersmall DG value. DG is greater at the fourth step, because of diminution of this enthalpyentropy compensation phenomenon. Thefour steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, andallosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal:mol when pHraises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of protonrelease (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately 13 kcal:mol is obtainedfor the successive steps of oxygenation (at pH 7.4, 37C). These results are compared with those previously obtained for pigeonand trout red blood cells. 1999 Elsevier Science Inc. All rights reserved.</p><p>Keywords: Bohr effect; Human hemoglobin; Human red blood cell; Oxygen affinity; Pigeon red blood cell; Temperature; Trout red blood cell;Vertebrate hemoglobin</p><p>www.elsevier.com:locate:cbpa</p><p>1. Introduction</p><p>Cooperative oxygen binding to hemoglobin (Hb) re-lies on transition from a deoxy structure (T) to an oxyone (R) [14]. The T-state is stabilized by ionic bonds,implying non-heme ligands such as protons and anionswhich are stepwise released upon oxygenation. The O2affinity of the T structure is lower than that of the Rone by the equivalent of the free energy of cooperativ-ity, of 3.6 kcal:mol under nearly physiological condi-tions for human HbA [15,16].</p><p>The thermodynamic characteristics of oxygenationfor human HbA solutions, based on the stepwiseAdairs scheme, have been examined [7,9,10,12,13]. Theintrinsic reactivity of the heme for oxygen is exothermicand constant, and the release of heterotropic ligands,namely H, Cl and 2,3 diphosphoglycerate (DPG)modulates the thermodynamics of the system. Measure-</p><p>ments of the oxygen equilibria at different temperaturesallow to estimate the enthalpy and entropy contribu-tions to the free energy of binding for each step ofoxygenation, and usefully complete equilibrium dataobtained at a single temperature.</p><p>Rather few data in the literature have examined theeffect of temperature on oxygen binding by humanblood, biochemically different and more complex thanthat of dilute Hb solutions, and essentially rely on thetemperature dependence of P50 [2,6,19,20]. Previousstudies have shown that the oxygen equilibria of nor-mal human red blood cells can be satisfactorily de-scribed by the Adair and MWC models [21,23]. In thepresent one, oxygen equilibria of human red blood cellshave been measured at five different temperatures from20 to 41C, and at eight pH levels from 6.8 to 8.2 inorder to vary the pH-induced constraints on the Hbmolecule. Results have been analysed according to theAdair and MWC models. They provide a description ofthe apparent thermodynamic properties of this system,* Tel.: 33-05-4944-4385; fax: 33-05-4944-4387.</p><p>1095-6433:99:$ - see front matter 1999 Elsevier Science Inc. All rights reserved.PII: S10 9 5 -6433 (99 )00068 -9</p></li><li><p>P. Vorger : Comparati6e Biochemistry and Physiology, Part A 123 (1999) 329336330</p><p>and may also constitute a basis of comparison forstudies on other Hb systems, since the temperaturedependence of oxygen binding by vertebrate Hbs isknown to exhibit large differences among species [3].</p><p>2. Materials and methods</p><p>All the experimental techniques used in this studyhave been previously described in detail [23,24]. Com-plete sets of oxygen equilibrium curves have beenmeasured on human red blood cells from normalhealthy adult donors, with automatic data acquisition.Blood was diluted in isotonic 0.05 M Tris or bisTrisbuffer0.125 M NaCl. The pH of the buffer wasadjusted at the exact temperature of that of measure-ments of the oxygen equilibria.</p><p>The Adair constants, ki (i14), MWC parame-ters (KT, the O2 association constant for the T-state,KR that for the R-state, c (KT:KR) and L (T0:R0), the allosteric constant) and overall O2 affinity,expressed as P50, were calculated by the method ofImai [8]. The enthalpy and entropy change for thesuccessive steps of oxygenation, i, were calculated bythe vant Hoff equations:</p><p>DHiRT2 d ln ki:dT</p><p>and</p><p>DSiR dT ln ki:dT,</p><p>where R and T are the gas constant and the absolutetemperature, respectively.</p><p>The average enthalpy and entropy change for over-all oxygenation, DHav and DSav, were obtained fromthe temperature dependence of ln P50.</p><p>The free energy change of ligand binding at thestep i is given by</p><p>DGiDHiTDSi.</p><p>The thermodynamic values for the MWC parame-ters were calculated from the temperature dependenceof ln L, ln KT, and ln KR.</p><p>3. Results and discussion</p><p>Examples of oxygen equilibria for human normalred blood cells, expressed as Hill plots, are presentedin Fig. 1, for the pH of 7.4 (unless specified, pHvalues are extracellular ones). Measurements wereperformed at the temperatures of 20, 25, 30, 37 and41C. The experimental data were fitted according tothe Adair equation. The binding curves are roughlyparallel to each other, indicating that the effect oftemperature is nearly independent of the level of oxy-genation, which rejoins early observations [25]. Thesame behavior applies to the other pHs examined(not shown). For the temperatures of 37 or 25C, thevalues of the Adair (or MWC) constants are similar,within the limits of experimental error, to those ob-tained in previous studies [21,23], and detailed valuesare not given here.</p><p>The vant Hoff plots for the temperature depen-dence of the Adair constants and P50, at pH 7.4, areshown in Fig. 2. The log ki and log P50 values havebeen plotted against 1:T. Assuming linearity for allthe parameters, the DH and DS values have beencalculated by linear regression.</p><p>Table 1 resumes the values of DH, DS, TDS andDG, obtained for the pHs of 6.8, 7.4, 7.8 and 8.0,and covering the pH range examined. At all pH lev-els, the enthalpies for the four steps of oxygenationappear to be nearly equal, with DH1DH2DH3DH4DHav. The standard errors are usually largerfor the two intermediate steps, as can be expectedfrom the plot of Fig. 2, due to the less good preci-sion for steps 2 and 3, which was also noted in otherstudies [9,12]. These heats include the heat of solutionof oxygen (3 kcal:mol), the intrinsic heat of oxygenbinding by the hemes, and all the heats associated tothe release of ions with oxygen binding with a majorrole of protons [5,13]. At each step, the reaction ismarkedly exothermic.</p><p>Fig. 1. Example of Hill plots for oxygen equilibria of human redblood cells diluted in 0.05 M Tris0.125 M NaCl buffer, at pH 7.4.Y, fractional oxygen saturation; P, partial oxygen pressure in mmHg.Temperatures are, from left to right: 20, 25, 30, 37 and 41C. Linescorrespond to experimental fit by the Adair equation.</p></li><li><p>P. Vorger : Comparati6e Biochemistry and Physiology, Part A 123 (1999) 329336 331</p><p>Fig. 2. Example of vant Hoff plots of the temperature dependence oflog ki (i14), and log P50 for human red blood cells at pH 7.4.T, absolute temperature. , k1; , k2; , k3; , k4; , P50. Linesare obtained by linear least-squares method.</p><p>thalpyentropy compensation process, as pointed outfor oxygen binding by human HbA solutions [7,10]. Asa result, the DG values are much smaller than the DHones. Furthermore, the uniformity observed for theDHs disappears, and the DGs exhibit the typical rela-tion: DG1DG2DG3BDG4.</p><p>Fig. 3 shows the compensation plot, where the valuesof DHi are plotted against those of DSi, for thevarious pHs examined. All the points corresponding toi1, 2 and 3 rather well lie on a straight line, calcu-lated by linear regression of these points, while thosefor i4 are clearly apart from that line. The compen-sation temperature, Tc, given by the reciprocal slope ofthe line, is 324 K, a value close to that of 300 Kobtained for human HbA solutions [7]. For the steps 1,2 and 3, the DH change is nearly counterbalanced bythat of DS, explaining the low value of DG. The in-crease of DG at the fourth step is the consequence of adiminution of the compensation process. This result isfully consistent with that obtained for HbA solutionsunder physiological conditions of pH and [DPG] [7]. Itcorresponds to the typical relation k15k2k3k4,also observed for human normal red blood cells [21].According to Perutz [15], the larger value of k4 is due tothe fact that the ionic bonds stabilizing the T-state havebeen broken during earlier steps of oxygenation.The changes of DSi are approximately parallel to</p><p>those of DHi. This can be expected from the vant Hoffequations, and corresponds to the well-known en-</p><p>Table 1Thermodynamic parameters for the oxygenation of human red bloodcells, at various pH levelsa</p><p>pH 8.0pH 7.8pH 7.4pH 6.8</p><p>11.291.7 10.291.0DH1 10.790.910.192.010.593.1DH2 6.192.7 11.095.7 11.096.3</p><p>8.893.2 8.494.4 9.792.7DH3 5.292.712.390.8DH4 13.990.811.692.5 11.890.6</p><p>DHav 11.491.410.791.59.390.89.490.7</p><p>30.195.7DS1 32.794.5 28.593.0 29.792.429.6910.3 13.698.3DS2 28.5918.5 28.5918.5</p><p>DS3 24.5910.614.798.6 25.198.725.395.2DS4 29.392.522.692.022.097.925.093.0</p><p>24.792.0 22.892.5DSav 26.594.7 28.394.8</p><p>9.991.4 8.790.9 9.090.7TDS1 9.191.79.093.1 4.192.5TDS2 8.795.6 8.795.6</p><p>TDS3 4.592.6 7.493.2 7.795.6 7.692.6TDS4 7.690.9 6.792.4 6.990.6 8.990.8</p><p>1.090.4DG1 1.390.2 1.590.3 1.790.31.590.8 2.092.0DG2 2.391.62.392.50.790.7 1.491.1DG3 0.790.8 2.191.3</p><p>DG4 4.790.8 4.992.8 4.990.7 5.090.7</p><p>a The values of DH, TDS and DG are in kcal:mol of oxygen, andthe DS values in cal:mol per deg. The standard state for DS is 1 atmof gaseous oxygen. DHav and DSav are averages of enthalpy andentropy changes for steps 14. Experimental conditions are as de-scribed in the text.</p><p>Fig. 3. Compensation plots of four-steps oxygen binding by humanred blood cells, for pHs 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 and 8.2. DSivalues are plotted against DHi values. For each given pH, the fouroxygenation steps are indicated by the same types. The line is drawnby linear least-squares method, through the points corresponding tosteps 1, 2 and 3. All the points clearly apart from that line correspondto step 4. Temperature compensation, Tc, as calculated from thereciprocal slope of the line, is 324 K.</p></li><li><p>P. Vorger : Comparati6e Biochemistry and Physiology, Part A 123 (1999) 329336332</p><p>Table 2Thermodynamic contributions to the free energy of cooperativity for human red blood cells oxygenation, at 37C and various pH levelsa</p><p>pH 7.4 pH 7.8 pH 8.0pH 6.8</p><p>3.2 (50%)T(DS4DS1) (kcal:mol) 1.8 (19%)1.5 (31%) 0.1 (16%)DH4DH1 (kcal:mol) 2.2 (27%) 0.4 (36%) 1.6 (15%) 3.2 (14%)</p><p>3.6 (86%) 3.4 (34%)DG4DG1 (kcal:mol) 3.3 (30%)3.7 (58%)</p><p>a All values are in kcal:mol of oxygen. The standard errors are indicated in parentheses.</p><p>Table 2 shows the enthalpy and entropy contribu-tions to cooperativity for oxygenation of human nor-mal red blood cells, expressed as the free energy change,DG4DG1, between the fourth and the first steps ofoxygenation. Results are rather homogeneous for allthe pHs examined, with DG4DG1 values ranging be-tween 3.7 and 3.3 kcal:mol. Although the esti-mates of DG4DG1 are affected by rather large errors, itappears that cooperative oxygenation of human redblood cells is not purely enthalpic in origin, but theconsequence of both enthalpic and entropicphenomena.</p><p>The present data have been also examined on thebasis of the MWC model. The temperature dependenceof log L at pH 7.4 is shown in Fig. 4 (the temperaturedependence of KT and KR is identical to that of k1 andk4 (Fig. 2), respectively). Calculations were done asdescribed by Imai [7], and results at pH 7.4 and 37Care shown in Table 3. At each step of oxygenation, theTR transition is endothermic. The allosteric quater-nary transition occurs at the third step, when DGitransbecomes negative, the entropic contribution exceedingthe enthalpic one. Roughly similar results were ob-tained for the other pHs (not shown). The allostericswitch can also be expressed by is ( log L:log c),the allosteric switchover [8]. At pH 7.4, is is equal to2.8. Similar values of 2.8, 3.0 and 2.6, i.e. close to 3,were obtained at pHs 6.8, 7.8 and 8.0, respectively.These results qualitatively agree with those for HbAsolutions [7]. They are consistent with the proposal byPerutz [16] that a fundamental event in the Hb</p><p>molecule occurs at the binding of the third oxygen.In Fig. 5, the DHav values have been plotted against</p><p>the pH. From pH 7.4 to 8.2, DHav augments by 2.8kcal, which can be attributed to a lower release of Bohrprotons at high pH, with diminution of their endother-mic contribution [1]. Below pH7.4, the DHav valueflattens. Although more difficult to explain, this resultrejoins the observation of larger heats at pH 6.5 than atpH 7.4, for HbA solutions [7]. At pH 7.4, the value of9.3 kcal:mol is in agreement with that of 8.2kcal:mol for human whole blood [19]. In the case ofintact red blood cells, as in the present study, it isassumed that the change of temperature has no effecton the pH difference between the extra and intracellularmilieus [18].</p><p>The values of the temperature coefficient, expressedas log P50:DT, are reported in Table 4. As DHav, thisparameter exhibits a slight pH-dependence, rangingfrom 0.022 at low pH to 0.028 at pH 8.2. At pH 7.4,the present value of 0.022 agrees with those of theliterature: 0.023 [19], 0.0226 [6] or 0.0229 [20].</p><p>A close relation is known to exist between the releaseof heat and the release of ions at the successive steps ofoxygenation [7,13]. It has been proposed [5] that therelease of Bohr protons would entirely account for theenthalpy change differences at the successive steps,according to the relation: DHiobsDH intDnDHH,</p><p>Table 3Thermodynamic parameters for allosteric TR transition of humanred blood cells, according to the MWC model, at 37C and pH 7.4,at successive oxgenation stepsa</p><p>Lci (Ti:Ri) TDSitrans DGitransDHitrans(kcal:mol)(kcal:mol)(kcal:mol)</p><p>T0R0 16106 8.7 1.3 10</p><p>T1R1 38.4103 8.3 1.9 6.4</p><p>2.65.37.9T2R2 0.9102</p><p>T3R3 7.52.2101 8.6 1.1</p><p>T4R4 4.811.95104 7.1</p><p>a DHitransDH0transi(DHTDHR), and DSitransDS0transi(DSTDSR). DH0trans and DS0trans are calculated from the temp...</p></li></ul>