Quantitative determination of carbamino adducts of alpha and beta chains in human adult hemoglobin in presence and absence of carbon monoxide and 2,3-diphosphoglycerate.

The principal component of normal adult human hemoglobin was equilibrated under various conditions with 13CO2. Quantitative analysis of the carbamino resonance intensities over the pH range of 6.5 to 9.0 shows that the effects of conversion from the deoxy to the liganded state in reducing the carbamino adduct formation occur predominantly at Val-1beta. Analysis of the pH dependence of carbamino formation at constant total carbonates yields values of pKz and pKc for Val-1beta and Val-1alpha in the deoxy and liganded conditions. In contrast to the Val-1beta as the allosteric site for CO2, the Val-1alpha site is shown to be primarily an alkaline Bohr group. 2,3-Diphosphoglycerate is shown to reduce substantially the Val-1beta carbamino resonance intensity in deoxyhemoglobin. Evidence for 2,3-diphosphoglycerate effects in carbon monoxide hemoglobin at both Val-1alpha and Val-1beta sites is presented. Enhanced carbamino formation in carbon monoxide hemoglobin at Val-1beta is observed at pH values less than 7.8. Finally, chemical exchange analysis of the spectra shows the release rate of the deoxy Val-1alpha carbamino adduct to be greater than that for deoxy Val-1beta. At pH 7.47 k-1obs,beta congruent to 1.0 and k-1obs, alpha congruent to 11.0 s-1.     

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)     

Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin.

The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.     

The acetylation state of human fetal hemoglobin modulates the strength of its subunit interactions: long-range effects and implications for histone interactions in the nucleosome

The source of the 70-fold increased tetramer strength of liganded fetal hemoglobin relative to that of adult hemoglobin between pH 6.0 and 7.5 reported earlier [Dumoulin et al. (1997) J. Biol. Chem. 272, 31326] has been identified as the N-terminal Gly residue of the gamma-chain, which is replaced by Val in adult hemoglobin. This was revealed by extending the study of the pH dependence of the tetramer-dimer equilibrium of these hemoglobins into the alkaline range as far as pH 9. From pH 7.5 to 9.0, the 70-fold difference in the association equilibrium constant between hemoglobins F and A lessened progressively. This behavior was attributed to the difference in the pK(a) 8.1 of Gly-1(gamma) compared to the pK(a) 7.1 value of Val-1(beta) of hemoglobins F and A, respectively. Evidence for this conclusion was obtained by demonstrating that natural hemoglobin F(1), which is specifically acetylated at Gly-1(gamma) and hence unable to be protonated, behaves like HbA and not HbF in its tetramer-dimer association properties over the pH range studied. An increased degree of protonation of the gamma-chain N-terminus of hemoglobin F from pH 9.0 to 8.0 is therefore suggested as responsible for its increased tetramer strength representing an example of transmission of a signal from its positively charged N-terminal tail to the distant subunit allosteric interface where the equilibrium constant is measured. An analogy is made between the effects of acetylation of the fetal hemoglobin tetramer on the strength of its subunit interactions and acetylation of some internal Lys residues within the N-terminal segments of the histone octamer around which DNA is wrapped in the nucleosome.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Hemoglobin providence. Functional consequences of two alterations of the 2,3-diphosphoglycerate binding site at position beta 82.

Position beta 82 in human hemoglobin (Hb) is normally occupied by lysine, a positively charged residue that is involved in the binding of anionic cofactors. This residue is substituted by a neutral residue in Hb Providence Asn and by a negatively charged residue in Hb Providence Asp. Hb Providence Asp shows more differences from Hb A than does Hb Providence Asn in studies of the kinetics and equilibria of ligand binding. For both forms, homotropic (cooperative) interactions are normal with n values of 2.5 to 2.7, while heterotropic (pH and anion) interactions are reduced greatly. The reduction in anion sensitivity is attributed to the absence of a positive residue at position beta 82. Reduction in pH sensitivity may be due to a ligand-linked change in the pK of a neighboring residue, beta 143 histidine, which normally is not a Bohr group. This change in pK would act in opposition to the normal Bohr effect. Reduction in the net positive charge of the central cavity has a further consequence. Relative to Hb A, both Hb Providence Asn and Hb Providence Asp show decreased oxygen affinities at neutral pH in the absence of cofactors. This suggests that in Hb A the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thus stabilizing the low affinity (T) conformation. From pH 6 to 9 in the presence of 1 M NaCl, where all the charged groups may be masked, the oxygen-binding properties of Hb A and the Hb Providence mutants are identical. Moreover, subunit dissociation of the liganded Hb Providence mutants appears to be increased, as is known to occur for Hb A in the presence of high salt. The results obtained with Hb Providence Asn and Hb Providence Asp illustrate how single amino acid substitutions can modify hemoglobins' pH and anion interactions without altering cooperative interactions between subunits. The alteration in cofactor effects observed with these mutants also illustrates differences between the allosteric effects induced by organic and inorganic anions.     

Hemoglobin of the Antarctic fishes Trematomus bernacchii and Trematomus newnesi: structural basis for the increased stability of the liganded tetramer relative to human hemoglobin

Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.     

Interactions between hemoglobin and organic phosphates investigated with 31P nuclear magnetic resonance spectroscopy and ultrafiltration.

1. The chemical shifts (delta) of the phosphates of 2,3-diphosphoglycerate and adenosine triphosphate (ATP) were determined by phosphorus nuclear magnetic resonance (31P NMR) spectroscopy and were found to be displaced downfield following the addition of hemoglobin (3 mM) to a solution of either diphosphoglycerate (5 mM) or ATP (1 mM). 2. The binding of these compounds to hemoglobin was also determined by membrane ultrafiltration. A direct relationship was observed between the change in chemical shift ((delta delta) of the 2-P and 3-P of diphosphoglycerate and the percent diphosphoglycerate bound, when the latter was varied by altering pH, oxygenation state, or total diphosphoglycerate concentration. 3. In comparable studies with ATP binding, a linear relationship between the delta delta values of the gamma-, beta-, and alpha-P of ATP and the percent of ATP bound was not observed when the data from all of the experiments were plotted. NMR signals were not detectible in deoxyhemoglobin solutions containing 1 mM ATP but were seen in solutions containing 3.8 mM ATP. 4. The results indicate that 31P NMR spectroscopy is a promising tool for investigating organic phosphate interactions with hemoglobin.     

Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding.

The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride.     

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y)

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.     

Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis

The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy-entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.     

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.     

Anion Bohr effect of human hemoglobin

The pH dependence of oxygen affinity of hemoglobin (Bohr effect) is due to ligand-linked pK shifts of ionizable groups. Attempt to identify these groups has produced controversial data and interpretations. In a further attempt to clarify the situation, we noticed that hemoglobin alkylated in its liganded form lost the Bohr effect while hemoglobin alkylated in its unliganded form showed the presence of a practically unmodified Bohr effect. In spite of this difference, analyses of the extent of alkylation of the two compounds failed to identify the presence of specific preferential alkylations. In particular, the alpha 1 valines and beta 146 histidines appeared to be alkylated to the same extent in the two proteins. Focusing our attention on the effect of the anions on the functional properties of hemoglobin, we measured the Bohr effect of untreated hemoglobin in buffers made with HEPES [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid], MES [2-(N-morpholino)ethanesulfonic acid], and MOPS [3-(N-morpholino)propanesulfonic acid], which being zwitterions do not need addition of chlorides or other anions for reaching the desired pH. The shape acquired by the Bohr effect curves, either as pH dependence of oxygen affinity or as pH dependence of protons exchanged with the solution, was irreconcilable with that of the Bohr effect curves in usual buffers. This indicated the relevance of solvent components in determining the functional properties of hemoglobin. A new thermodynamic model is proposed for the Bohr effect that includes the interaction of hemoglobin with solvent components. The classic proton Bohr effect is a special case of the new theory.     

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins

An allosteric modulator of oxygen release in human erythrocytes is 2,3-diphosphoglycerate, but bovine erythrocytes apparently utilize chloride for this purpose since they contain little, if any, 2,3-diphosphoglycerate. In order to identify the sites to which these anions bind, the site-specific acetylating agent, methyl acetyl phosphate, has been employed to compete with these allosteric modulators and to mimic their effects on hemoglobin function. With human hemoglobin A, methyl acetyl phosphate competes with 2,3-diphosphoglycerate and acetylates only Val-1(beta), Lys-82(beta), and Lys-144(beta) within or near the cleft that binds this organic phosphate (Ueno, H., Pospischil, M. A., Manning, J. M., and Kluger, R. (1986) Arch Biochem. Biophys. 244, 795). With bovine hemoglobin, the acetylation is competitive with chloride ion. The sites of acetylation in oxy bovine hemoglobin are Met-1(beta) and Lys-81(beta) and for deoxy bovine hemoglobin, they are Val-1(alpha) and Lys-81(beta). Thus, these sites are expected to be involved in the binding of chloride to bovine hemoglobin. Treatment of either human or bovine hemoglobins with methyl acetyl phosphate under anaerobic conditions leads to a lowering of their oxygen affinity and hence the covalent modifier has the same effect on hemoglobin function as the non-covalent regulators, 2,3-diphosphoglycerate and chloride. The Hill's coefficient of hemoglobin is unaffected by treatment with methyl acetyl phosphate. Under aerobic conditions, specifically acetylated bovine hemoglobin also has a lowered oxygen affinity, and human hemoglobin A shows a slight change in its oxygen affinity. In general, bovine hemoglobin is more responsive than human hemoglobin to both chloride and methyl acetyl phosphate; the latter agent results in a permanent covalent labeling of the protein. Therefore, the results support the idea that methyl acetyl phosphate may be a useful probe for deciphering the sites of binding of anions to proteins.     

31P-NMR investigation of trimethylphosphine binding to [alpha Fe(II), beta Mn(II)] hybrid hemoglobin. A model for partially liganded species

31P-NMR of trimethylphosphine binding to the ferrous chains of a ([alpha Fe(II), beta Mn(II)]hemoglobin hybrid is employed to investigate partially liganded species. This study shows that at low pH (6.5), in the presence of inositol hexaphosphate, the resonance at 23.2 ppm (from H3PO4) is due to phosphine bonding to alpha-chains in the T quaternary state. At elevated pH (7.6), phosphine binding to the alpha-chains produces a resonance at 24.8 ppm which is associated with a T-to-R conversion. These findings are discussed in relation with our previous results on direct observation of intermediate ligation states of hemoglobin.