Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.     

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.     

On the oxygen-linked anion-binding sites in human hemoglobin. Functional properties of human hemoglobin reacted with 4-isothiocyanatobenzenesulphonic acid and its hybrids

Human hemoglobin, reacted at the four amino termini with 4-isothiocyanatobenzenesulphonic acid (Hb-ICBS), was separated into its constituent chains. Recombination of the ICBS-reacted chains with the unmodified mate chains produced the hybrid tetramers modified at either the beta or the alpha chains: alpha 2 beta 2ICBS and alpha 2ICBS beta 2. All of the modified tetramers show a reduced oxygen affinity and reduced cooperativity; furthermore the oxygen affinity of the Hb-ICBS and alpha 2 beta 2ICBS is unaffected by 2,3-bisphosphoglycerate while the oxygen affinity of alpha 2ICBS beta 2 is decreased in the presence of this organic phosphate. The oxygen affinity of Hb-ICBS and alpha 2ICBS beta 2 is independent of chloride concentration, while the alpha 2 beta 2ICBS hybrid shows a reduced response to this anion. The tetramers alpha 2ICBS beta 2 and alpha 2ICBS beta 2ICBS show a decreased alkaline Bohr effect, which can be rationalized as being due to disruption of the oxygen-linked chloride-binding sites; in the case of alpha 2 beta 2ICBS the Bohr effect is instead (partially) maintained. The functional properties of artificial tetramers have been studied also from a kinetic point of view by CO combination and the results obtained compare satisfactorily with equilibrium data. The possibility of obtaining selectively modified hemoglobins promises to provide further insight into the properties of the oxygen-linked anion-binding sites in hemoglobin.     

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.     

Allosteric transition in triply met-haemoglobin

Methaemoglobin undergoes a transition to a T-like form at acid pH in the presence of strong effectors such as inositol hexakisphosphate (IHP), as evidenced by spectroscopic and oxidation potential measurements. Since oxygen and CO do not bind to the ferric haems, it is difficult to compare the properties of the R-met and T-met forms with those of ferrous haemoglobin. We have therefore prepared 90% oxidized samples, where the dominant signal for ligand (oxygen or CO) binding is due to tetramers with three met haems. Measurements were made of the oxygen equilibrium curves and CO rebinding kinetics after photodissociation. Without effectors, the partially oxidized samples show mainly R-state properties. Addition of IHP at acid pH induces an increase in T-state behaviour, as indicated by a lower oxygen affinity and a higher fraction of the slow bimolecular component for CO rebinding.     

Redox Bohr effects and the role of heme a in the proton pump of bovine heart cytochrome c oxidase

Structural and functional observations are reviewed which provide evidence for a central role of redox Bohr effect linked to the low-spin heme a in the proton pump of bovine heart cytochrome c oxidase. Data on the membrane sidedness of Bohr protons linked to anaerobic oxido-reduction of the individual metal centers in the liposome reconstituted oxidase are analysed. Redox Bohr protons coupled to anaerobic oxido-reduction of heme a (and Cu(A)) and Cu(B) exhibit membrane vectoriality, i.e. protons are taken up from the inner space upon reduction of these centers and released in the outer space upon their oxidation. Redox Bohr protons coupled to anaerobic oxido-reduction of heme a(3) do not, on the contrary, exhibit vectorial nature: protons are exchanged only with the outer space. A model of the proton pump of the oxidase, in which redox Bohr protons linked to the low-spin heme a play a central role, is described. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

A calorimetric study of the CO Bohr effect of monomeric haemoglobins.

A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups. A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4 JK-1 mol-1) at pH 9.5 as compared to pH 6.0.     

Subunit hybridization studies of partially ligated cyanomethemoglobins using a cryogenic method. Evidence for three allosteric states

Reaction of tetrameric hemoglobin with ligands at the four heme sites yields nine species that have structurally unique combinations of ligated and unligated subunits. Using hemoglobins where the ligated subunits contain cyanomethemoglobin, Smith and Ackers studied the dimer-tetramer assembly reactions in all nine of the partially ligated species (F. R. Smith and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 5347). They found a third assembly free energy in addition to those of unligated hemoglobin and fully ligated cyanomethemoglobin. The observed distribution of the three assembly free energies among the ten species was found to be incompatible with the two-state mechanism of allosteric control (J. Monod, J. Wyman and J. P. Changeaux, J. Mol. Biol. 12 (1965) 81). The results indicated a mechanism of 'combinatorial switching' in which the binding free energies per site change with configuration of occupied sites and not just their number. In this study, we have confirmed the existence of three assembly free energies among the ten ligation species using a cryogenic method (M. Perrella and L. Rossi-Bernardi, Methods Enzymol. 76 (1981) 133). For one of the species we find a different free energy assignment from that reported by Smith and Ackers; for all other species we observe the same assignments as in earlier work. The revised distribution also requires a 'combinatorial' mechanism of allosteric switching among the three states.     

Preparation and characterization of dimeric bovine hemoglobin tetramers

Dimeric bovine hemoglobin (Hb) tetramers were prepared by a one-step solid phase adsorption method. Briefly, Hb was absorbed by the solid phase, Q Sepharose Fast Flow media, followed by reaction with the glutaraldehyde and elution procedure. Then, dimeric bovine Hb tetramers were formed and purified from Hb tetramers by anion-exchange chromatography based on Protein-Pak DEAE 8HR. The dimeric Hb tetramer showed a P50 value of 15.9 mm Hg, oxygen transporting efficiency of 14.2%, and Hill coefficient of 1.72. The number of Bohr protons released for dimeric Hb tetramers was 0.59 H/tetramer, which was 39% of that of native bovine Hb. The number of chloride ions released on oxygenation was 0.60/tetramer for dimeric Hb tetramers, which was 46% of that of native bovine Hb.     

Single-site modifications of half-ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer

The patterns of energetic response elicited by single-site hemoglobin mutations and chemical mocdifications have been determined in order to probe the dimer–dimer interface of the half-ligated tetramer (species[21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules¹. In this study the free energies of quaternary assembly(dimers to tetramers) were determined for aseries of 24 tetrameric species in which one dimeric half-molecule is ligated while the adjacent αβ dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half-ligated tetramers and the unligated tetramers over the entire spatial distrubution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer–dimer interface of the half-ligated tetramer(species[21]) has the same quaternary sturcture as that of the unligated molecule, i.e, “quaternary T.” This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T → R quaternary switchpoints². Analysis of the observed Symmetry Rule behaviour in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between α and β subunits of the dimeric half-tetramer within quaternary T. The mutational results indicate that these interactions are not “paid for” by breaking or making noncovalent bonds at the dimer–dimer interface (α¹β²). They arise from structural and energetic changes that are “internal” to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of “tertiary constraint” is thus generated by the first binding step and is propagated to the second hemesite while the dimer–dimer interface α¹β²serves as a constraint. The “sequential” cooperativity that occurs within the half-molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers. When the constraint is released functionally by T to R dimer rearrangement (at each switch-point specified by the a Symmetry Rule) the alterations of interfacial bonds then dominate the energetics of cooperativity. © 1993 Wiley-Liss, Inc.     

Hagfish hemoglobins: structure, function, and oxygen-linked association

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528).     

Ligand-dependent Bohr effect of Chrionomus hemoglobins.

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation. For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2-liganded and CO-liganded states of hemoglobin III. Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi; here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively. On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine-E11, obstructing the movement of the heme-iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved. The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.     

Source of residual Bohr effect in hemoglobin oxidation.

The hemoglobin oxidation Bohr effect is larger than the ligation Bohr effect, even when the former is corrected for any ionization of the water molecule bound to the ferric iron of methemoglobin. This residual oxidation Bohr effect is here shown to be solely caused by the influence of the positively charged ferriheme, and is abolished when the oxidized heme binds an anion. This result frees the formal equivalence of hemoglobin ligation and oxidation from the last apparent experimental discrepancy.     

The dissociation of carbon monoxide from the alpha and the beta subunits of human carbonmonoxy hemoglobin

We have measured the intrinsic CO dissociation rates from the subunits of the human hemoglobin tetramers (alpha CO beta NO)2 and (alpha NO beta CO)2 using microperoxidase and a stopped-flow spectrophotometer. The dissociation of NO is negligible. The rate constants for the and the subunits are similar (0.014 s-1 vs. 0.011 s-1, respectively, at pH 7, 20 C; and 0.016 s-1 for both in the presence of inositol hexaphosphate), indicating that they are equivalent in the first step of the CO dissociation. Therefore, the chain unequality observed in the third and fourth steps (Samaja, M., Rovida, E., Niggeler, M., Perrella, M., and Rossi-Bernardi, L. (1987). J. Biol. Chem.: 262, 4528-4533) are not due to the intrinsic properties of the subunits, but to the conformational state of the molecule.     

PH dependence of the Adair constants of human hemoglobin. Nonuniform contribution of successive oxygen bindings to the alkaline Bohr effect.

In order to solve the problem of an apparent discrepancy between the pH variance of oxygen equilibrium curve and the linear relation between the number of released Bohr protons and the degree of ligation, precise oxygen equilibrium curves of human hemoglobin were determined at a number of pH values from 6.5 to 8.8. From the equilibrium data individual steps (Adair constants), ki (i equals 1, 2, 3, 4), were obtained and the number of Bohr protons (deltaHi+) released on the ith stage of oxygenation was estimated. The pH dependence of k4 was very small, while the other ks strongly depended on pH over the pH range examined. As a consequence, the contribution of each step of oxygen binding to the alkaline Bohr effect nonuniform: deltaH4 was very small compared with deltaH1+, deltaH2+, and deltaH3+. In spite of this, calcuation has shown that the fractional number of released protons is essentially proportional to fractional oxygen saturation because of cooperative effects in hemoglobin. Thus, the present study indicates that the linear relationship between the fractional number of released protons and the degree of ligation, as obtained from titration experiments, is not necessarily incompatible with the pH variance of the shape of the oxygen equilibrium curve. The nonuniform pH depencence of the Adair constants implies that the two-state allosteric model of Monod, J., Wyman, J., and Changeus, J.P. (1965) J. Mol. Biol. 12, 88-118 is not adequate to describe the heterotropic effect caused by protons.     

Energetics of subunit assembly and ligand binding in human hemoglobin.

An extensive and self-consistent set of thermodynamic properties has recently been established for the coupled processes of subunit assembly and ligand binding (oxygen and protons) in human hemoglobin. The resulting thermodynamic values permit a consideration of the possible sources of energetic terms accounting for stability of the tetrameric quaternary structures at different stages of ligation, and of the possible sources of cooperative energy. The analysis indicates that: (a) The change in buried surface ara upon oxygenation (i.e., hydrophobic stabilization) does not play a dominant role in stabilizing the unliganded tetramer relative to the liganded tetramer. (b) The pattern of enthalpic and entropic contributions to the free energies of dimer-tetramer. (c) The thermodynamic results are consistent with a dominant role of increased hydrogen bond formation in the deoxy quaternary structure. (d) Within tetramers the variation in free energy for successive oxygenation steps arises from both enthalpic and entropic contributions and the enthalpic contributions are almost entirely attributable to the heats of Bohr proton release. At pH 7.4 the pattern of thermodynamic values suggests that a large contribution to the free energy of cooperativity may arise from the energetics of Bohr proton release. It is suggested that a combination of proton ionization and hydrogen bonding may account for the main energetic features of cooperativity. Possible contributions from fluctuation behavior cannot presently be evaluated.     

Properties of carboxymethylated cross-linked hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and comparison of arylsulfatase A from rat liver and Morris hepatoma 7777

Rat liver and Morris hepatoma 7777 arylsulfatase A were isolated from the soluble lysosomal extract by a procedure involving blue-Sepharose affinity chromatography, DEAE-cellulose chromatography, hydrophobic chromatography on phenyl-Sepharose and preparative polyacrylamide gel electrophoresis. The preparation obtained by this method was apparently homogenous in disc electrophoresis and in immunoelectrophoresis. The comparative studies revealed that the properties of arylsulfatase A from rat liver and Morris hepatoma 7777 are very similar, considering molecular weight of the native monomer and its subunits, the ability to form tetramers, isoelectric point, Michaelis constant and the anomalous kinetics of the reaction. The twofold elevation of arylsulfatase B activity found in Morris hepatoma 7777 suggests that the enzyme may have certain functions in tumor growth.     

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structural and functional analysis of the hemoglobin D component from chicken

Oxygen binding by chicken blood shows enhanced cooperativity at high levels of oxygen saturation. This implies that deoxy hemoglobin tetramers self-associate. The crystal structure of an R-state form of chicken hemoglobin D has been solved to 2.3-A resolution using molecular replacement phases derived from human oxyhemoglobin. The model consists of an alpha2 beta2 tetramer in the asymmetric unit and has been refined to a R-factor of 0.222 (R-free = 0.257) for 29,702 reflections between 10.0- and 2.3-A resolution. Chicken Hb D differs most from human oxyhemoglobin in the AB and GH corners of the alpha subunits and the EF corner of the beta subunits. Reanalysis of published oxygen binding data for chicken Hbs shows that both chicken Hb A and Hb D possess enhanced cooperativity in vitro when inositol hexaphosphate is present. The electrostatic surface potential for a calculated model of chicken deoxy-Hb D tetramers shows a pronounced hydrophobic patch that involves parts of the D and E helices of the beta subunits. This hydrophobic patch is a promising candidate for a tetramer-tetramer interface that could regulate oxygen binding via the distal histidine.