Linkage of organic phosphates to oxygen binding in human hemoglobin at high concentrations

We have performed high-precision oxygen binding studies on human hemoglobin tetramers in the presence of a series of limited, subsaturating amounts of the effector compounds 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP). The use of thin-layer optical methods enabled the use of high hemoglobin concentrations, preventing complications arising from the dissociation of the tetramer into dimers. Model-independent, simultaneous analysis of all data for each effector demonstrated that the intrinsic oxygen binding characteristics of the molecule are in agreement with those determined in earlier high-precision studies [e.g., Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry 26, 3995-4002] and that the affinity of the tetramer for the tightly binding effector IHP changes most markedly between the second and fourth oxygen binding steps, perhaps indicating a large conformational change. The data were then analyzed by using the truncated allosteric model [Di Cera, E., Robert, C. H., & Gill, S. J. (1987) Biochemistry 26, 4003-4008], which is based on the hypothesis that a quaternary conformational change occurs in the hemoglobin tetramer before the third and fourth oxygen molecules bind.     

Allosteric interpretation of the oxygen-binding reaction of human hemoglobin tetramers

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.     

Precision determination and Adair scheme analysis of oxygen equilibrium curves of concentrated hemoglobin solution. A strict examination of Adair constant evaluation methods

To examine the validity of the recent finding by Gill et al. (S.J. Gill, E. Di Cera, M.L. Doyle, G.A. Bishop and C.H. Robert, Biochemistry 26 (1987) 3995) that the third overall Adair constant (A3) for human hemoglobin tetramers (Hb A) is too small to be determined and therefore that the contribution of the triply ligated species in the oxygenation process is negligibly small, highly accurate oxygen equilibrium curves for concentrated pure Hb A solutions were determined with an automatic oxygenation apparatus and analyzed by a least-squares curve-fitting method with various options. The present results indicate that an appropriate choice of weighting for data points is the key to the correct evaluation of the Adair constants and the present experimental data cannot accommodate the Adair scheme with A3 = 0, giving distinctly positive values for A3. Several criteria for correct determination of the Adair constants are presented.     

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., and Robert, C. H. (1987) Biochemistry, 26, 3995-4002), the heats for binding two and four oxygen molecules were found to be simple multiples of the first binding heat. A direct consequence of equal stepwise heats is invariance of the shape of the binding curve with temperature, as pointed out by Wyman (Wyman, J. (1939) J. Biol. Chem. 127, 581-599). Titration microcalorimetry was also performed for the binding of carbon monoxide to hemoglobin. While the tight binding of CO precludes high-precision binding measurements, it does allow one to accurately determine the heat of ligation as a function of the CO bound. In these titrations a uniform heat of reaction is not observed, but the heat of binding increases markedly near the end point. This implies that the stepwise binding enthalpy for adding the third CO molecule is anomalously endothermic and for adding the fourth strongly exothermic. A similar phenomenon cannot be ruled out in the case of oxygen because of imprecision intrinsic in the analysis of the weaker ligand binding.     

Nested allosteric interaction in tarantula hemocyanin revealed through the binding of oxygen and carbon monoxide

We have examined the competitive binding of oxygen and carbon monoxide to the multisubunit hemocyanin of the tarantula Eurypelma californicum. Employment of high-precision thin-layer methods has enabled detailed characterization of the pure oxygen and pure carbon monoxide binding curves, as well as binding curves performed under mixed-gas conditions. The pure oxygen binding curve and the displacement of oxygen by carbon monoxide at full ligand saturation are highly cooperative, but in the absence of oxygen, carbon monoxide binds noncooperatively. The results were analyzed globally within the framework of a nested allosteric model [Robert, C.H., Decker, H., Richey, B., Gill, S.J., & Wyman, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1891-1895] which takes into account the hierarchy of subunit structure present in the macromolecule. The use of two ligands enables one to recognize two distinct levels of allosteric interaction functioning in the protein assembly. The binding characteristics of the allosteric states demonstrated for Eurypelma follow a similar pattern as those found earlier for Homarus americanus.     

New twists on an old story: hemoglobin

New determinations of oxygen and carbon monoxide binding to human hemoglobin, indicating an extremely small population of triply ligated species, suggest a close relationship with structural features that govern the allosteric mechanism of cooperativity.     

Kinetic investigations of the quaternary enhancement effect and alpha/beta differences in binding the last oxygen to hemoglobin tetramers and dimers

Analysis of O2 binding equilibria by two independent groups has suggested that the affinity for binding the fourth O2 to Hb tetramers is very high, about 800-1200 cal/mol higher than that of dimers (Chu, A. H., Turner, B. W., and Ackers, G. K. (1984) Biochemistry 23, 604-167; Di Cera, E., Robert, C. H., and Gill, S. J. (1987) Biochemistry 26, 4003-4008). Recently, Gibson and Edelstein challenged the reality of the quaternary enhancement effect, based on kinetic data (Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516-519). However, these studies failed to directly address the key issue of the relative affinities of dimers and alpha 2 beta 2(O2)3. Furthermore, the extent to which alpha/beta differences influence these results remains an open question. Using partial laser photolysis and O2/CO replacement techniques we have, for the first time, resolved the rates of O2 association and dissociation to both alpha and beta chains within "R state" tetramers and dimers. We find that the beta chains are faster than alpha for both O2 binding (approximately 2-fold) and release (approximately 3-fold). The kinetically determined O2 affinities derived from these data are essentially identical for dimers and alpha 2 beta 2(O2)3. That is, the data do not show significant quaternary enhancement and suggest that the equilibrium data have both overestimated the affinity of alpha 2 beta 2(O2)3 and underestimated the affinity of dimers. The significance of and possible origins for the discrepancy between equilibrium and kinetic data are discussed.     

Oxygen binding constants for human hemoglobin tetramers

High-precision studies of oxygen binding in hemoglobin (HbA0) solutions at near-physiological concentrations (2-12 mM heme; pHs 7.0-9.1; various buffers) have led to an unanticipated result: an unmeasurably low contribution from the triply ligated species. We have obtained this result from new differential oxygen-binding measurements for human hemoglobin through the use of a thin-layer apparatus, which enables study of solutions at high Hb concentrations. The effect of tetramer dissociation into dimers, which becomes significant at hemoglobin concentrations below 1 mM in heme, is avoided. The analysis of the binding reactions is thus cast in terms of tetramer-binding polynomial written with overall Adair equilibrium constants which directly reflect the contributions of intermediate ligated species. The unmeasurable contribution of the triply ligated species renders the equilibrium constants of the third and fourth stepwise reactions practically undeterminable.     

Ligand-controlled dissociation of Chromatium vinosum cytochrome c'

Carbon monoxide binding to Chromatium vinosum ferrocytochrome c' has been studied by high-precision equilibrium methods. In contrast to the CO binding properties of Rhodospirillum molischianum cytochrome c' [Doyle, M. L., Weber, P. C., & Gill, S. J. (1985) Biochemistry 24, 1987-1991], CO binding to C. vinosum cytochrome c' is found to be unusual in the following ways. The binding curve is found to be cooperative with typical Hill coefficients equal to 1.25. The shape of the binding curve is asymmetrical. The heat of CO ligation is measured by two independent methods, both of which yield large endothermic values of approximately 10 kcal [mol of CO(aq)]-1. The overall affinity for CO increases as the concentration of cytochrome c' decreases. These observations suggest the CO binding properties of C. vinosum cytochrome c' are complicated by CO-linked association-dissociation processes. Further investigation by gel filtration chromatography shows that at micromolar concentrations the dimeric state is tightly associated in both the reduced and oxidized forms of the cytochrome but addition of saturating concentrations of CO causes the reduced ligated dimer to dissociate largely into monomers. A model is presented that quantitatively fits the data, involving a ligand-linked dimer-monomer dissociation reaction. In this model, CO binds to the dimer form noncooperatively with an intrinsic affinity constant equal to 5600 +/- 1200 M-1 at 25 degrees C. The unligated dimer form is tightly associated, but addition of CO causes dissociation of the dimer into the monomer with a monomer-dimer association constant equal to 450 +/- 200 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

An analysis of oxygen binding by trout (Salmo gairdneri) red cells

• 1.1. Oxygen equilibrium curves have been measured on ATP-depleted trout red cells, at pHs between approx. 7 and 9, at temperatures of 20 and 15°C.
• 2.2. Results were analysed according to the model of Di Cera E., Robert C. H. and Gill S.J. (Biochemistry, 1987 26, 4003–4008), using a non-linear least-squares method. This allosteric model, proposed for human HbA, assumes the existence of two quaternary states, T and R, plus large functional differences between the α and β chains within the T state.
• 3.3. For this material, the fit was found rather better than that previously observed with the original Monod-Wyman-Changeux model. This is in accord with the idea that important α-β chain differences, a demonstrated property of Root effect Hbs, would play an important role in oxygen binding by trout red cells, where the Root effect Hbs are dominant.
• 4.4. In comparison, the fit was found poor for oxygen binding by human red cells, suggesting that the hypothesis of important chain differences would not apply to this material.

     

Oxygen binding constants and stepwise enthalpies for human and bovine hemoglobin at pH 7.6.

A high-precision thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand-binding macromolecules in a manner analogous to that of the Gill thin-layer optical apparatus [Doleman & Gill (1976) Anal. Biochem. 87, 127]. We have generated differential heat-binding curves of oxygen binding to human and bovine hemoglobin in phosphate buffer at pH 7.6, with the enzyme-reducing system of Hayashi et al. [(1973) Biochim. Biophys. Acta 310, 309]. Experiments were conducted at a number of different temperatures in order to expand the data field, allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumptions of optical linearity between the various heme groups and reveals that the triply ligated species is measurably significant for both human and bovine hemoglobin. It was also determined that the concentration of doubly ligated species of bovine hemoglobin is relatively low. The experiments indicate that the reactions for both hemoglobins are enthalpy-driven for oxygen stepwise additions 1, 2, and 4 while being entropy-driven for step 3. Human hemoglobin oxygen-binding experiments were also performed with the Gill thin-layer optical apparatus under solution conditions identical to those used in the calorimeter. The experiments revealed that if optical linearity is assumed, the overall third equilibrium constant is negative or near zero. This indicated that either the optical cell's performance is much poorer than the thin-layer calorimeter or there is an appreciable nonlinear optical effect.     

Description of the molecular mechanism of cooperativity in human hemoglobin cannot be limited to a first-order free energy coupling concept

Studies of the linkage between ligand binding and subunit assembly of oligomeric proteins have extensively used the concept of free energy coupling. The "order" of these free energy couplings was introduced [Weber, G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7098-7102] as the number of subunits that must be liganded to alter specific intersubunit interactions. This concept dictates that the ligation of fewer subunits has no effect, but once the order number of subunits becomes ligated, the specific intersubunit interaction energy between those particular subunits is completely eliminated. Weber's report claims that the free energy coupling between oxygen binding and the dimer-tetramer subunit assembly in stripped human hemoglobin A is "first order". This conclusion is based on the analysis of a set of previously published equilibrium constants [Mills, F. C., Johnson, M. L., & Ackers, G. K. (1976) Biochemistry 15, 5350-5362]. I subsequently reported that the original experimental data, from which the equilibrium constants were derived, are consistent with both the first-order and "second-order" free energy coupling concepts [Johnson, M. L. (1986) Biochemistry 25, 791-797]. I also demonstrated that more precise recent experimental data [Chu, A. H., Turner, B. W., & Ackers, G. K. (1984) Biochemistry, 23, 604-617] are consistent with both the first-order and second-order free energy coupling concepts. A recent article [Weber, G. (1987) Biochemistry 26, 331-332] disagrees that the oxygen-binding data for human hemoglobin A are consistent with a second-order model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of linkage in macromolecules when one ligand is present in limited total quantity

We present a general framework for analysis of two closely related problems in biochemical studies: (1) The first is analysis of binding data obtained under conditions in which a second, linked ligand is present in limited total quantity. In such conditions the free activity of the second ligand varies throughout the primary ligand binding curve, and the resultant behavior can be quite complex. Analysis of such curves enables one to quantitatively extract detailed information regarding the linkage of the two ligands at intermediate stages of ligation. The treatment is applied in an accompanying paper to oxygen binding in human hemoglobin in the presence of organic phosphates [Robert, C.H., Fall, L., & Gill, S. J. (1988) Biochemistry (following paper in this issue)]. (2) The second treatment we outline regards the analogous problem of analyzing differential scanning calorimetry (DSC) data obtained for a macromolecule binding a ligand present in limited quantity. A simple model is presented that accounts for dual transitions like those already seen in DSC data for human serum albumin in the presence of nonsaturating amounts of fatty acids [Ross, P., & Shrake, A. (1987) Abstracts of the 42nd Calorimetry Conference, University of Colorado, Boulder, CO].     

Carbon dioxide and oxygen linkage in human hemoglobin tetramers

Differential binding curve measurements for oxygen in the presence of fixed carbon dioxide activities have allowed a detailed determination of the linkage between carbon dioxide and the oxygenated intermediates of human hemoglobin. Model-independent analysis of the data shows that at pH 7.4: (1) the oxygen binding curves are asymmetrical, the population of the triply oxygenated species being negligible; (2) the shape of the oxygen binding curve is invariant with carbon dioxide activity; (3) the maximum linkage is -0.32 moles carbon dioxide per mole oxygen; and (4) the overall carbon dioxide-dependent shift in the oxygen binding curve cannot be explained in terms of carbamino formation alone, the additional influence of bicarbonate being required. An allosteric model that accounts for the low population of triply oxygenated hemoglobin species is employed here as a framework from which to explore the carbon dioxide linkage mechanism at the intermediate stages of oxygenation. Carbon dioxide binding constants are found to be 780 M-1 and 580 M-1 for carbon dioxide binding to the deoxygenated alpha and beta chains, respectively, and 150 M-1 for carbon dioxide binding to the oxygenated form of both chains, as determined by simultaneous fitting of the oxygen binding curves with the model. Finally, by use of the determined binding polynomial for the carbon dioxide-oxygen linkage scheme, we have constructed a series of linkage graphs.     

Cooperative free energies for nested allosteric models as applied to human hemoglobin.

A model is developed for ligand binding to human hemoglobin that describes the detailed cooperative free-energies for each of the ten different ligated (cyanomet) species as observed by Smith and Ackers (Smith, F.R., and G.K. Ackers. 1985. Proc. Natl. Acad. Sci. USA.82:5347-5351). The approach taken here is an application of the general principle of hierarchical levels of allosteric control, or nesting, as suggested by Wyman (Wyman, J. 1972. Curr. Top. Cell. Reg. 6:207-223). The model is an extension of the simple two-state MWC model (Monod, J., J. Wyman, and J.P. Changeux. 1965. J. Mol. Biol. 12:88-118) using the idea of cooperative binding within the T (deoxy) form of the macromolecule, and has recently been described as a "cooperon" model (Di Cera, E. 1985. Ph.D. thesis). The T-state cooperative binding is described using simple interaction rules first devised by Pauling (Pauling, L. 1935. Proc. Natl. Acad. Sci. USA. 21:186-191). In this application three parameters suffice to describe the cooperative free-energies of the 10 ligated species of cyanomet hemoglobin. The redox process in the presence of cyanide, represented as a Hill plot, is simulated from Smith and Ackers' cooperative free-energies and is compared with available electrochemical binding measurements.     

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.     

Inhibition of fibrinogen binding to human platelets by blockage of Na+/H+ exchange

Binding of ADP to platelets enhances the binding of fibrinogen to Gp IIb-IIIa, the specific platelet receptor for adhesive proteins. The linkage between ADP and fibrinogen binding is indirect since ADP does not bind to the same receptor as fibrinogen. We have recently proposed that a third component, once affected by ADP binding, induces a conformational transition of the fibrinogen receptor from the low to the high affinity state, which is responsible for platelet aggregation [De Cristofaro, R., Landolfi, R., Castagnola, M., De Candia, E., Di Cera, E., & Wyman, J. (1988) Proc. Natl. Acad. Sci. USA 85, 8473-8476]. In the present study we provide evidence that this component should be identified with the platelet Na+/H+ antiport. Inhibition of the antiport by pharmacological agents such as amiloride, or else by decreasing extracellular Na+, results in a marked decrease of fibrinogen binding to platelets.     

Stabilization of the T-state of hemoglobin

The effect of inositol hexaphosphate and bezafibrate on binding of O2 and CO to HbAO at high concentrations (1 mM) has been evaluated using thin layer optical techniques. Data analysis shows 1) the occurrence of greatly reduced ligand dependent cooperativity (Hill slope of 2.23 for CO and 1.51 for O2), and 2) the presence of significant triply ligated species. The data fits a nested allosteric two-state MWC model in which the T state consists of two allosteric substrates, Tt and Tr, where Tt binds only to the alpha chains and Tr binds to both alpha and beta chains. The model indicates that the triply ligated species consists of a predominant amount of T form, agreeing with kinetic observations of CO ligated hemoglobin. The maximum amount of triply ligated R molecules (CO or O2) implicated is less than 1%, a result similar to that found previously for binding studies made in the absence of BZF and IHP.     

Binding of oxygen and carbon monoxide to arthropod hemocyanin: an allosteric analysis

The binding of oxygen and carbon monoxide to hemocyanin from the mangrove crab Scylla serrata and the lobster Homarus americanus has been studied by thin-layer optical absorption and front face fluorescence techniques. Three types of experiments were performed on subunit and oligomeric preparations of each hemocyanin: oxygen binding, carbon monoxide binding, and oxygen-carbon monoxide competition studies. The results obtained from the subunit preparations of dissociated oligomers from both hemocyanins show that the binding site can be ligated by either one oxygen or one carbon monoxide. The binding results obtained with the oligomeric samples of hemocyanin from both species cannot be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118] since the data from the three types of binding experiments cannot be fit with a single set of binding constants. The MWC model has been extended by including a third allosteric form, and an analysis based on the three-state model is able to fit the data from the three types of experiments with the same set of binding constants. The comparison of the oxygen to carbon monoxide affinity ratios (kO2/kCO) indicates that the structure around the binding site of subunits in the T form oligomer is similar to that of the free subunits. The oligomeric forms of both these hemocyanins bind carbon monoxide with a weak but definite positive cooperativity. An analysis of the affinity ratios for the T, S, and R forms suggests that the high affinity of the R form results from a specific interaction between oxygen and binding site.