In 1904, BOHR, HASSEL$ALCH, and KROGH

Japanese Journal of Physiology, 34, 205-216, 1984

MINIREVIEW

The Bohr Effect and the Haldane Effect in Human Hemoglobin

Itiro TYUMA

Department of Physicochemical Physiology, Medical School, Osaka University, Osaka, Osaka, 530 Japan

In 1904, BOHR, HASSEL$ALCH,and KROGH [11] discovered that increased carbon dioxide pressure (Pcoz) shifts the oxygen equilibrium curve of blood to the right, i.e., the oxygen affinity of blood is inversely proportional to Pcoz. There was much discussion at that time as to whether the shift due to C02 could be explained entirely by the concomitant change in pH, or whether C02 has, in addition, a specific effect. It has now been shown that C02 does exert a specific effect due to its direct combination with hemoglobin to form carbamino compound [27]. Thus, the original observation of Bohr et al., now called "classical Bohr effect" [19, 39], is a composite of the specific effects of C02 and Ht The separate effect of pH on the oxygen affinity of hemoglobin has conventionally been referred to as the Bohr effect : between pH 6 and 9 the fall of pH decreases the oxygen affinity (the alkaline Bohr effect) and below pH 6 the oxygen affinity rises with falling pH (the acid or reverse Bohr effect). On the other hand, the reduction of C02 content in blood on oxygenation at constant Pcoz was demonstrated by CHISTIANSEN,DOUGLAS, and HALDANE[13] in 1914. This "classical Haldane effect" [39] is a thermodynamic corollary of the classical Bohr effect and can be explained by the decrease in carbamino binding to hemoglobin and the fall in pH on oxygenation. The release and uptake of protons on oxygenation and deoxygenation of hemoglobin are now defined as the Haldane effect. Sometimes both the Bohr and Haldane effects, including the classical ones, are collectively termed the Bohr effect. It is now well-established that in addition to protons and carbon dioxide, 2, 3-diphosphoglycerate (DPG), the major organic phosphate in mammalian erythrocytes, tremendously decreases the oxygen affinity of hemoglobin by binding to the deoxy form of the protein preferentially [9]. Other salt anions, e.g., chloride and inorganic phosphate, also show a similar but lesser effect [24]. Although the binding sites of these ligands are remote from the heme iron atom, the bindings are related to the oxygenation of the iron atom and have been described as "oxygen-linked" or "oxylabile." The interactions between the oxygen-binding sites and the sites for the non-heme ligands are referred to as the heterotropic allosteric

Received for publication December 28, 1983 中馬一郎

205 206 I. TYUMA

effects or interactions [32]. The bindings of the non-heme ligands are also mutually related [28], as will be shown below. The present review deals with the thermodynamic relation, molecular mechanism, and physiological significance of the Bohr and Haldane effects in human hemoglobin. For excellent historical reviews see ROUGHTON[38] and EDSALL [16].

THERMODYNAMICALRELATION BETWEENTHE BOHR EFFECT AND THE HALDANE EFFECT General linkage relations When a macromolecule binds two or more different ligands, e.g., oxygen and proton, there is an interdependence of the bindings due to interaction between the binding sites: the reactivity of a site is affected by the presence of a ligand at another site in the same molecule. When this occurs, the bindings are called mutually "linked." The linked phenomena can be analyzed in terms of general thermodynamic relations independent of the mechanism involved. This was first done by ADAIR[1] and successfully developed by WYMAN[44, 45]. Let us consider the binding equilibria of two different ligands, X and Y, with hemoglobin that has q binding sites for X and r sites for Y. One of the linkage relations is described by the following equation [44] : ax aY 1 q aln Y )=r_-) x alnx () y where X and Y are the fractional saturation of hemoglobin molecule with X and Y, and x and y are their activities. Equation (1) states that if the binding of X influences the binding of Y, the binding of Y must also influence the binding of X. Among many other linkage relations derived from Eq. (1), the following one is very useful to describe the heterotropic interactions:

q (ax _ (&lny\ r aY x a In x Y' (2) Apparently Eqs. (1) and (2) are valid in the presence of any third component only if its activity is independent of oxygenation. This qualification is very important when one deals with the classical Bohr and Haldane effectswhich involve three different kinds of ligand, i.e., 02, H+, and C02.

The Bohr and Haldane coefficients Letting X be H+ and Y be 02, Eq. (2) becomes

(aH+ \ _ ( a logP02 a Y )pH.- apH \ Y' (3) where H+ is the number of protons bound per heme and Y is fractional oxygen

Japanese Journal of Physiology BOHR AND HALDANE EFFECTS 207

saturation. As mentioned above, the left-hand side of Eq. (3) gives the magnitude of the Haldane effect and is therefore called the Haldane coefficient, whereas the right-hand side is referred to as the Bohr coefficient. Thus, thermodynamically the Bohr and Haldane coefficients are equivalent. If the shape of the oxygen equilibrium curve (Y versus log F02) i s independent of pH, Eq. (3) can be transformed into a more convenient form, i.e.,

JH+= J JHlog P50 (4) p where JH+ is the increase in the number of protons (per heme) bound to hemoglobin upon full oxygenation and £50 is Pot at Y=0.5. Equation (4), which has conventionally been used, states the equivalence of the Bohr coefficient at Y=0.5 and the average value of the Haldane coefficient for the various oxygen satura- tions between 0 and 1. JH+ can be directly determined by measuring the change in pH which ac- companies the combination of deoxyhemoglobin with oxygen at desired pH values and back-titrating to the original pH values. The right-hand side of Eq. (4) is obtained from the slope of the log F50 versus pH plot. The relation for human adult hemoglobin (Hb A) is graphically represented in Fig. 1 [2]. Protons are released (4H+ is negative) and taken up (JH+ is positive) upon oxygenation above pH 6 and below pH 6, respectively. These oxygen-linked protons are called Bohr protons. The value of 4H+ for Hb A at 20°C in the absence of carbon dioxide and DPG is about -0.6 at pH 7.3 [10], and it decreases numerically with increasing Pco2 and temperature. pH dependence of the shape of oxygen equilibrium curve The assumption made in deriving Eq. (4), i.e., the pH invariance of the shape of the oxygen equilibrium curve is thermodynamically equivalent to stating that

Fig. 1. The Bohr and Haldane effects on Hb A at 20°C. Dependence of log P50 (upper panel) and 4W (lower panel) upon pH. Ionic strength 0.2 to 0.4 M [2].

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Fig. 2. Experimental equivalence of the Bohr (0) and Haldane coefficients (t) of Hb A. A, pH 7.65; B, pH 8.48. Temperature: 30°C. Broken lines show the average values of both the coefficients. the release of protons is proportional to oxygenation. This has long been ac- cepted as one of the characteristic features of mammalian hemoglobins [4, 44]. In the last decade, however, the invalidity of the assumption, in a strict sense, was shown by several investigators [20, 23, 26, 40, 42]. The available bodies of experimental evidence show that the upper part of the oxygen equilibrium curve is less sensitive to pH change than the other part : the magnitude of the Bohr coefficient depends upon Y. It is now established that the fourth oxygen binding constant, k4, is insensitive to pH change, i.e., the binding of the last oxygen molecule does not release protons [23, 24]. Numerical inequality of the Bohr coefficient at Y=0.5 and the average value of the Haldane coefficient was shown by SIGGAARD-ANDSERSENet al. [40]. In consistent with these findings, the simultaneous determination of proton release and oxygen saturation performed on Hb A have clearly shown that the fractional number of protons liberated is not strictly proportional to the fractional oxygen saturation [43]. The determination, moreover, allowed us to compare the Bohr and Haldane coefficients at various oxygen saturation [43]. As il- lustrated in Fig. 2, both the coefficients are dependent on Y near their extremes and agree quite well with each other in a full range of Y : slight discrepancies ob- served at low P02 and Y are within experimental errors. Thus, the original equation (3) has now been experimentally substantiated. The validity of (aH~/ log Poz)'pH'=(aY/apH)'Poz', which is derived from Eq. (1), has also been sup- ported by the simultaneous measurement of proton release and oxygenation [43].

Japanese Journal of Physiology BOHR AND HALDANE EFFECTS 209

The classical Bohr and Haldane coefficients In human whole blood at the standard condition, i.e., plasma pH=7.40 (red cell pH= 7.20), Pco 2=40 mmHg, total DPG concentration [DPG] = 5 mM, at 37°C, the components that affect oxygen affinity of hemoglobin are H+, C02, and DPG; the effects of other anions will be ignored here for the sake of simplicity and their lesser importance as regulators of gas transport. The classical Bohr coefficient can be defined as the change in log Pot due to the change in pH which is accomplished solely by a change in Pco2, and described as follows [40] : log Pot a log Pot apH )Y ,[DPG]- apH )Y,Pco2,{DPG] + a log Pot (&logPco2\ 5 a log Pc0 Y H DPG apH Y DPG, ( ) The first term of the right-hand side of Eq. (5) expresses the change in oxygen affinity with pH at constant Pc02 and total DPG concentration (the Bohr effect). The second term indicates the specific effect of C02 on the oxygen affinity : less carbamino compound is formed by oxyhemoglobin than by deoxyhemoglobin (oxygen-linked carbamino binding). The reported values for the classical Bohr coefficient at the standard state are in the range of -0.50 to -0.54, and the first and last terms of the right-hand side of Eq. (5) are determined to be -0.35 and -0 .15, respectively [19]. Physiologically, the classical Bohr coefficient is important because in vivo the pH is mainly determined by Pco2. The coefficients are also dependent upon Y, and their values at different Y are reported by GARBY et al. [19] and HLASTALAand WoonsoN [21]. ARTURsoNet al. [5] have tabulated the values, for whole blood, of the coefficients d log Pot/d pH (plasma), 4 log P02/4 log [DPG], and d log Pot/ J log Pco2 at different values of Y, plasma pH, Pco2, and total DPG concentration. When comparing data for whole blood with that obtained from hemoglobin solution, the difference in pH between red cells and plasma should be considered. The correction for differences in pH can be made by the following relation :

( a a log X \ - ( a log X apH (cell) 6 pH (plasma) Y apH (cell) Y apH (plasma) Y ' ( ) Under physiological conditions, the numerical value of (a pH (cell)/a pH (plasma))'Y' is 0.80 to 0.84 and it varies with pH, Y, Pco2, and [DPG] [18]. It is important to note that Eq. (5) is written in terms of total concentration of DPG (free and hemoglobin bound) instead of the activity of DPG. This is done for practical purposes since it is almost impossible to maintain constant DPG activity during oxygen equilibrium measurement. Theoretically, therefore, one cannot apply the linkage relationships, e.g., Eq. (3), to the coefficients described in Eq. (5). Under normal physiological conditions, however, changes in the

Vol. 34, No. 2, 1984 210 I. TYUMA

DPGactivity seem to be unimportantas longas the total DPGconcentration is constant[18]. The classical Haldane coefficient can be definedas the changein totalcarbon dioxideper heme,[C02], due to the changein oxygensaturation at constantPc0 2, and expressedas follows[40] : a[C02] aH+ a[C02] aY )PCO2,[DPG]aY )pH,PCO2,[DPG] aH ~r,PC02,[DPG] + (a[C02]aY (7 ) c02, pH, [DPGJ The first term of the right-hand side of Eq. (7) describes the change of [C02] due to the change in acidity on oxygenation (the Haldane effect). The second term shows oxygen-linked carbamino binding of hemoglobin. The numerical values for the classical Haldane coefficient in the standard state are reported to be about -0 .28, of which about two thirds is due to the first term and the remaining one third is attributable to the second term [7, 29]. Evidently, the classical Bohr and Haldane effects are not equivalent.

MOLECULARMECHANISM OF THE BOHR EFFECT X-ray crystallographic studies by PERUTZand his associates [34] have clearly indicated that the hemoglobin molecule undergoes a reversible structural transition when oxygen is bound or removed. In deoxy form the molecule is in a constrained conformation with a low oxygen affinity (T-state, T for tense), whereas in oxy form it assumes a relaxed conformation where the oxygen affinity is high (R-state, R for relaxed). The physiologically important allosteric effects of hemoglobin are now explained by the change of reactivity of binding sites for ligands accompanied by the T-R transition. WYMAN[44] showed that the Bohr effect can be described by postulating that each subunit of hemoglobin has two oxygen-linked acid groups (the Bohr groups); the pK of one group increases and the pK of the other decreases upon oxygenation. The former group is responsible for the acid Bohr effect and the latter for the alkaline Bohr effect. X-ray crystallographic and other studies, however, have shown that the alkaline Bohr effect cannot be ascribed to only one acid group, and consists of contributions of several Bohr groups. Their pK values and relative contributions in the absence of organic phosphates are listed in Table 1. In the T-state a-amino group of 1a Val and imidazole group of 146,3 His form salt bridges between carboxyl groups of 141a Arg and 94 Asp, respectively, thereby increasing their pK values. On oxygenation the salt bridges are broken due to the structural transition to R-state, decreasing the pK values, and the Bohr groups dissociate protons [36]. At present there is no straightforward interpreta- tion for the pK change of 122a His at the molecular level. The unexplained 20

Japanese Journal of Physiology BOHR AND HALDANE EFFECTS 211

Table 1. Contribution of the individual Bohr groups to the alkaline Bohr effect and their pK values at 25°C and pH 7.4 in the absence of organic phosphates.

portion was attributed to the oxygen-linked binding of chloride ions which in- volves uptake of protons [28, 35]. Carbon dioxide binds to the uncharged N-terminal a-amino groups, with higher affinity for T-state than for R-state, releasing protons. The released protons compensate for the uptake of protons by the other Bohr groups on deoxygenation, descreasing the magnitude of the Bohr effect. In the presence of organic phosphates, all the positively charged groups at the DPG binding site, i.e., 1j3Val, 2/3 His, and 14313His, are involved in the alkaline Bohr effect since the DPG binding is accompanied by H+ uptake [35]. Thus, the Bohr coefficient is significantly increased (numerically) by DPG [l0]. The alkaline Bohr effect strongly depends on the heterotetrameric nature of hemoglobin: isolated a and j3 chains show little or no Bohr effect, although the latter chains exist in a tetrameric form, /34[30, 41]. The molecular mechanism of the acid Bohr effect is still uncertain.

PHYSIOLOGICAL SIGNIFICANCE OF THE CLASSICAL BOHR AND HALDANE EFFECTS

The physiological role of the classical Bohr and Haldane effects has generally been characterized by theoretical approaches rather than experimentation. It is difficult to design an experiment to block the classical Bohr effect in vivo.

Significance in the transport of oxygen As clearly pointed out by the discoverers, the classical Bohr effect will show little or no influence on oxygen transport in the lung, because the mixed-venous- arterial difference in pH and Pcoz is small and the upper part of the oxygen equilibrium curve is relatively insensitive to changes in both pH and Pcoz [23, 25]. On the other hand, in tissues with a high metabolic rate, the classical Bohr effect is of physiological significance. During a submaximal exercise of about 200 W, the Pot and Pcoz in the mixed venous blood are about 20 and 65 mmHg (base excess -11.05 mEq/liter), respectively, and the pH averages 7.164 [15]. BAYER[7] calculated that in this case the arterio-mixed venous difference in oxygen saturation will be 75 . If

Vol. 34, No. 2, 1984 212 I. TYUMA there is no classical Bohr effect and the pH remains 7.414, the arterio-mixed venous difference will be 62 %. Thus, the classical Bohr effect provides about 20 % of the amount of oxygen made available to the working muscle. In the placenta, the maternal and fetal blood exchanges oxygen and carbon dioxide: the former becomes more acid and the latter more alkaline. Hence, the classical Bohr effect is twice as effective. In the maternal blood oxygen is released more readily, since the oxygen affinity decreases, and the fetal blood takes up oxygen more easily because of its high oxygen affinity. BARTELSestimated that the classical Bohr effect may contribute by 45 % to the transfer of oxygen from maternal to fetal blood near term [6]. Quantitative consideration based on a mathematical model shows, however, that the Bohr effect accounts for only about 8 % of the oxygen exchange across the placenta [22].

Significance in the transport of carbon dioxide With a normal respiratory exchange ratio (R.Q.) of 0.85, 0.85 mol of C02 are given up in the lungs per mol of oxygen uptake. As mentioned already, the value for the classical Haldane coefficient is about 0.28 mol C02/mol 02. There- fore, the contribution of the classical Haldane effect to total exchange of carbon dioxide is 0.28/0.85, or 33 %. The remainder of C02 exchange is accomplished by a decrease in dissolved C02 with resultant changes in hydrogen ion and bicarbonate concentration. Apparently the contribution is dependent upon the value of R.Q. and becomes greater in alveoli with low R.Q. There was sharp disagreement on the relative importance of the chemical mechanisms involved in the classical Haldane effect, i.e., the relative contributions of the first and second term of the right-hand side of Eq. (7) to the total effect. WYMAN[44] suggested that the pH difference between oxy- and deoxyhemoglobin solutions might be sufficient to account for the extra C02 contained in the deoxyhemoglobin solutions without the need to suppose that the carbamino reactions of hemoglobin are oxygen-linked. On this basis, he thought that carbaminohemoglobin plays only a minor role in physiological C02 transport, the Haldane effect (the first term) being capable of negotiating essentially all of the oxygen-linked transport. ROUGHTON[37], on the other hand, concluded that about 70% of the classical Haldane effect can be explained by the decrease in carbamino binding to hemoglobin on oxygenation. After that it was shown that DPG binding to hemoglobin and carbamino formation are antagonistic processes and that this should be taken into account in assessing the role of carbaminohemoglobin in the classical Haldane effect. Careful studies of BAUERand SCHROEDER[8] eventually indicated that at the standard condition the fraction of oxy-labile carbamate is 0.092 mol C02/ mol 02, and that the contribution of carbaminohemoglobin to the classical Haldane effect is therefore about 33 %. Contribution to the total C02 transport is 11%.

Japanese Journal of Physiology BOHR AND HALDANE EFFECTS 213

According to the mathematical model of HILL et al. [22], the classical Haldane effect accounts for about 46 % of the C02 exchanged in the placenta.

Kinetic limitations The classical Bohr and Haldane effects must proceed to completion in the time course of capillary transit (about 1 sec) to be of physiological value. MAREN and SWENSON[31] measured the velocity of the displacement of oxygen from the red cell suspension on the sudden addition of C02 (the Bohr shift or the Bohr-off reaction), and the half-time was determined as 0.12 sec at physiological conditions, sufficient to yield equilibrium within the average capillary transit time. Addi- tion of 10_3 M methazolamide, a carbonic anhydrase inhibitor, increased the half- time to 4.2 sec. When fixed acid was added to the suspension, the half-time increased to 0.32 sec. The acid instantly reacts with HC03- to give H2C03, which dissociates into C02 and water, and CO2 diffuses into the red cell where it is catalytically hydrated to yield HC03- and protons for reaction with hemoglobin. The slow rate of the uncatalyzed dehydration of H2C03 is responsible for the increased half-time. These results agree quite well with those reported by FORSTERand his colleagues [14, 17]. The kinetics of the Haldane shift (or the Bohr-on reaction) was estimated by measuring the rate of the release of carbon dioxide from the red cell suspension on the sudden increase of Pot [29]. The half-time of Pco, change was 0.144 sec, which is also within the capillary transit time. On the other hand, the change of oxygen saturation of hemoglobin was completed within 0.1 sec, indicating that diffusion of oxygen inside the cell and reaction of hemoglobin with 02 are not rate-limiting. Movement of 02 and C02 through the red cell membrane is also not likely a limiting factor, since these molecules are uncharged and lipid soluble, which enhances erythrocyte permeability. Release of Bohr protons and the combination of these protons with bicarbonate to form carbonic acid are also unlikely to limit the rate of Bohr and Haldane shifts : the release of Bohr protons occurs within a few msec [3], and the latter ionic reaction is essentially instantane- ous. Thus, in the presence of an excess of carbonic anhydrase, the most likely rate-limiting step is the transmembrane exchange of chloride and bicarbonate, the half-time of which has been reported to be 0.09 sec [12]. When carbonic anhydrase is completely inhibited, carbon dioxide hydration becomes rate-limiting and the rate of the Bohr shift is reduced to abolish its occurrence within capillary transit.

SUMMARY

Protons and carbon dioxide are physiological regulators for the oxygen affinity of hemoglobin. The heterotropic allosteric interaction between the non-heme ligands and oxygen, collectively called the Bohr effect, facilitates not only the transport of oxygen but also the exchange of carbon dioxide. Several

Vol. 34, No. 2, 1984 214 I. TYUMA types of interactions can be thermodynamically formulated. The Bohr and Haldane coefficients and the classical Bohr and Haldane coefficients are thus explicitly defined, which will save confusion about the use of the term "Bohr effect" seen in the literature. Molecular mechanism and the physiological significance of the classical Bohr and Haldane effects are outlined. The latter effect seems to play a far greater physiological role than the reciprocal influence of carbon dioxide on oxygen transport the classical Bohr effect.

Key Words: Bohr effect, hemoglobin, oxygen equilibrium, Haldane effect, carbon dioxide.

REFERENCES

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