The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices

Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 m-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146)β. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94)β, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 m-NaCl + 0.2 m-phosphate this change of ligation lowered the pK_a of the histidine from 8.0 in Hb³ to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pK_a in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect.We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)β → Leu), HbCO Wood (His FG4(97)β → Leu) and HbCO Malmø (His FG4(97)β → Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)β in fact belongs to His FG4(97)β. Although in Hb the pK_a of His HC3(146)β is 8.05 ± 0.05 independent of ionic strength, in HbCO its pK_a drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pK_a of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973).In HbCO A, His FG4(97)β does not contribute to the Bohr effect, but in HbCO from which His HC3(146)β has been cleaved (HbCO des-His), His FG4(97)β is in equilibrium between two conformations with different pK_a values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety.In HbCO A, His FG4(97)β has a pK_a of 7.8 compared to the pK_a value of about 6.6 characteristic of free histidines at the surface of proteins. This high pK_a is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of α-helices, which manifests itself even when they lie on the surface of the protein.     

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.     

Alteration of functional properties associated with the change in quaternary structure in unliganded haemoglobin.

The preparation of a number of specifically modified haemoglobins lacking various C-terminal residues is described. These haemoglobins can be changed from the unliganded R (or oxy type) quaternary structure to the unliganded T (or deoxy type) on addition of inositol hexaphosphate. This paper shows that this transition is associated with a lowering of the oxygen affinity and an increase in the Hill's coefficient, n, except in the case of des-(Arg141α, Tyr140α) haemoglobin where addition of inositol hexaphosphate lowers the oxygen affinity but does not increase the Hill's coefficient, n. This shows that Tyr140α plays a more important role than Tyr145β in generating co-operativity. The transition between unliganded R and unliganded T is associated with a lowering of the reactivity of the sulphydryl group Cys93β; this is due both to the change in quaternary structure per se and to the formation of the salt bridge between His146β and Asp94β. The Bohr effect associated with the transition from the unliganded to liganded R structure was less than one-tenth of the normal Bohr effect.     

Differences between horse and human haemoglobins in effects of organic and inorganic anions on oxygen binding.

Despite the fact that the horse is one of the more common domesticated animals, there are few reports dealing with the properties of its blood, and no comprehensive study has been performed on the reactivity of horse haemoglobin towards organic and inorganic ions. Here we report data on the effects of the organic phosphates D-glycerate-2,3-bisphosphate (2,3-DPG) and InsP6, and of chloride on the properties of horse haemoglobin. Thus the effect of saturating concentrations of 2,3-DPG on the oxygen affinity of horse haemoglobin is about 60% lower than with human adult haemoglobin under the same experimental conditions. The same applies also to InsP6, whose effect on oxygen binding to horse haemoglobin is decreased by about 55% compared with human adult haemoglobin. On the whole, horse haemoglobin appears to be much less sensitive to organic phosphates than previously believed. These results are discussed in the light of the primary structure of the molecule.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

Effect of 2,3-diphosphoglycerate on the Bohr effect of human adult hemoglobin

The effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect of human hemoglobin has been studied by means of hydrogen ion titration techniques. The results indicate a) that both the acid and the alkaline Bohr effect are equally affected, b) that the DPG binding to deoxyhemoglobin (Hb) is much stronger than to carboxyhemoglobin (HbCO) and c) that Hb binds effectively one DPG molecule. The effect on the Bohr effect can roughly be described by assuming that upon binding two groups per tetramer change their pK from 6.8 to 7.8 and two others from 6.8 to 5.8. These groups very probably are the imidazole groups of the two histidines H21 (143)β and the two phosphate groups of DPG (second dissociation). From the experiments a value for the dissociation constant K of the Hb-DPG complex of about 10⁻⁵ M⁻¹ could be estimated at pH 6.2 and pH 7.5.     

2,3-Diphosphoglycerate Content of Human Arterial and Venous Blood

THE glycolytic intermediate, 2,3-diphosphoglycerate, is an intracellular regulator of the oxygen affinity of haemoglobin^1,2. At high altitudes there is a direct relationship between the decreased oxygen affinity of haemoglobin and the increased concentration of diphosphoglycerate in the blood³. This was explained by Benesch et al.⁴ and Chanutin et al.⁵, who found that the binding of diphosphoglycerate to haemoglobin reduces the oxygen affinity and by our finding that the concentration of diphosphoglycerate increases when the red cells are incubated under low oxygen tension^6,7, thereby releasing oxygen from haemoglobin. For the same reason, the oxygen tension is reduced during the circulation of blood from the pulmonary alveoli to the tissues; the decreased level of the diphosphoglycerate facilitates the binding of oxygen to haemoglobin in the pulmonary alveoli and the increased level of the diphosphoglycerate in the blood of the capillaries decreases the affinity of haemoglobin for oxygen. We have measured the amount of 2,3-diphosphoglycerate and other glycolytic intermediates in arterial and venous blood to test this supposition.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Effects of Temperature, pH, and Organic Phosphates on Fish Hemoglobins

This review presents data on effects of temperature, pH, and organic phosphates on structural-functional characteristics of fish hemoglobins. Special attention is paid to the structure of pigments insensitive to pH as well as those that have the reverse Bohr effect in alkaline range of pH. The role of surface -adrenoreceptors and activity of Na⁺/H⁺ exchanger in regulation of the acid-base equilibrium of intraerythrocyte medium are considered. There are compared characteristics of participation of nucleotide triphosphates, 2,3-DPG, and other organic phosphates in correction of fish hemoglobin affinity to oxygen. The values of H for fish respiratory pigments are presented, and their dependence on conditions of erythrocyte microsurrounding is discussed.     

The oxygen-linked zinc-binding site of human haemoglobin.

Zn2+ is known to increase the 02 affinity of human haemoglobin. Previous data suggested that Zn2+ exerts its effect by directly binding to haemoglobin, rather than by competing with or binding to 2,3-bisphosphoglycerate. It was also shown that there are two 02-linked zinc-binding sites in haemoglobin, and that Zn2+ does not significantly alter haemoglobin co-operativity or the alkaline Bohr effect. The effect of Zn2+ on 02 affinity of haemoglobin can also be observed for other haemoglobins as diverse as those of cow and chicken. This paper presents new data on the haemoglobin-zinc interaction for normal haemoglobin, des-His146beta-haemoglobin and N-ethylsuccinimide-haemoglobin of humans. For normal haemoglobin (0.05 mM in tetramers), at 20 degrees C in buffer containing 0.1 M-Cl-, 02-dissociation-curve experiments showed that the addition of 0.4-0.5 mM-ZnS04 did not change the Bohr effect between pH 6.71 and 7.29. Similar experiments, with "zinc-ion buffers", showed that the value of the Hill coefficient, h, decreased only slightly if the concentration of free Zn2+ was held constant. For N-ethylsuccinimide-haemoglobin, Zn2+ caused less increase in O2 affinity than for normal haemoglobin. These studies, together with data on the equilibrium binding of Zn2+ to oxy-, deoxy- and des-His146beta-haemoglobins, suggest that zinc is chelated in oxyhaemoglobin by at least three amino acids, two of which are histidine-146beta and cysteine-93beta.     

Influence of anions and protons on the Adair coefficients of haemoglobins A and Cowtown (His HC3(146) beta----Leu)

We have measured the contribution of the alkaline Bohr effect of the C-terminal histidine residues of the beta-chains of haemoglobin A by comparing haemoglobin A with haemoglobin Cowtown in which those histidine residues are replaced by leucine. Oxygenation of a stripped 2.5 mM (haem) solution of haemoglobin A yielded 0.19 H+/haem, while oxygenation of a similar solution of haemoglobin Cowtown produced no change of pH. Oxygen equilibria measured at 60 microM-haem in 0.1 M-Hepes buffer gave an alkaline Bohr effect of -0.21 H+/haem for haemoglobin A and only -0.01 H+/haem for haemoglobin Cowtown, even though its Hill's coefficient was greater than 2 throughout the pH range studied. These results prove that the chloride-independent part of the alkaline Bohr effect is due to the C-terminal histidine residues of the beta-chains. Oxygen equilibria measured in 0.095 M-bis-Tris buffers with minimal chloride or with 0.1 M-chloride showed the contribution of those histidine residues to the alkaline Bohr effect to be about 0.2 H+/haem, independent of chloride concentration. Determination of the individual Adair coefficients in the three different buffers indicated that pH and chloride tend to have their greatest effects at the second or third steps of oxygenation when the change of quaternary structure is most likely to occur; between pH 7 and 9, the fourth Adair coefficient is only very slightly affected by pH and not significantly by chloride.     

Adaptation to Hypoxia by Increased HbO<Subscript>2</Subscript> Affinity and Decreased Red Cell ATP Concentration

THERE is a decrease in the O₂ affinity of mammalian haemoglobin (Hb) as the levels of 2,3-DPG or ATP are increased, which is explained by an allosteric effect on the HbO₂ binding^1,2. Similar observations on amphibians³ and fish⁴, which have molar ratios of ATP to Hb similar to those of DPG to Hb in mammals, suggest that red cell organic phosphates modulate Hb function in all vertebrates. The adaptation of mammals to various hypoxic stresses involves reduced HbO₂ affinity^5–9, the attendant increase in O₂ “unloading” capacity being mediated by an increase in the concentration of red cell 2,3-DPG. We have found the opposite response in hypoxic fish and suggest that an increased O₂ affinity results in increased O₂ transport for the fish.     

Postnatal changes in 2,3-diphosphoglycerate (2,3-DPG) content of calf erythrocytes

In 82 calves, 22 adult cows and 10 fetuses the 2,3-DPG level was determined in the erythrocytes. The lowest level was found in cows (1.13 microM/g haemoglobin, on the average), and in calves aged 35 days or more. In the foetuses the mean 2,3-DPG concentration in the erythrocytes was 4.98 microM/g Hb and it was higher that that in the erythrocytes of cows, the oldest foetuses and calves during the first two days of postnatal life. During 5 weeks of postnatal life the changes taking place in 2,3-DPG concentration could be divided into two periods: period I or the period of increase covering the first 6 days of life, with a characteristic rise in the concentration of this component from 1.37 to 15.80 microM/g Hb, and period II or the period of decrease lasting from the 6th to the 35th day of life. In period II two phases could be discerned, the first phase lasting from the 6th to the 10th day with a steep fall of 2,3-DPG level from 15.80 to 4.58 microM/g Hb, and the second phase from the 10th to the 35th day of life in which the level of 2,3-DPG reached slowly the value found in adult cows. A comparison of oxygen affinity of haemoglobin in calves aged 6 days, which was composed of about 80% of fetal haemoglobin and about 20% of adult haemoglobin, and in adult cows, which contained exclusively adult haemoglobin, showed that the oxygen-binding capacity of haemoglobin was lower in calves.     

Comparison of carbonic anhydrase activity and other haematological values of two Eutherian and three Australian marsupial mammals

1. Carbonic anhydrase activity and 2,3-diphosphoglycerate (2,3-DPG) concentration were determined in whole blood from humans (Homo sapiens), rabbits (Oryctolagus cuniculus), eastern grey kangaroos (Macropus giganteus), pademelons (Thylogale billardierii) and brush-tailed possums (Trichosurus vulpecula). 2. Marsupial blood carbonic anhydrase activity increased as species body size decreased. 3. T. billardierii haemoglobin was found to have a polymorphism which may be the same (beta 2 = histidine or glutamine) as that of M. giganteus. 4. The concentration of 2,3-DPG int e red cells of T. billardierii was approximately equal to that of the haemoglobin tetramer. Levels of 2,3-DPG in the other species were similar to those previously reported.     

Age-related changes in haemoglobin-oxygen affinity and haematology of rats at high altitude

• 1.1. Exposure of 4-, 12- and 40-week old rats to a simulated altitude of 5000m elicited an increase in 2,3-DPG and a concomitant decrease in the oxygen affinity of the red blood cells.
• 2.2. Differences in 2,3-DPG and P₅₀ between the three age groups which had been apparent at sea level were not significant after 12hr at high altitude.
• 3.3. All age groups showed marked increases in haemoglobin concentration and haematocrit at high altitude although the increases were less marked for the 4-week old group after increases due to post-natal development were subtracted.

     

Histidine proton resonances of carbonmonoxyhaemoglobins A and Cowtown in chloride-free buffer

A re-examination of the C-2 histidine proton resonances of haemoglobins A and Cowtown (His HC3(146)β → Leu) in chloride-free Hepes buffer has shown that all the resonances present in haemoglobin A are present in haemoglobin Cowtown, so that the pK_a of His HC3(146)β cannot be determined by nuclear magnetic resonance in this buffer.     

2,3-Diphosphoglycerate affects Enzymes of Glucose Metabolism in Red Blood Cells

THE bulk of organic phosphate in the erythrocyte exists in the form of 2,3-diphosphoglycerate (2,3-DPG), which is known to determine the position of the oxygen dissociation curve^1,2 as well as to influence the glycolytic pathway as a cofactor of the monophosphoglyceromutase (MPGM) reaction³ and as an inhibitor of its own formation in the diphosphoglyceromutase reaction⁴. But whether 2,3-DPG inhibits the hexokinase reaction is unclear and reports are contradictory^5–7.     

Hemoglobin Warsaw (Phe beta 42(CD1)----Val), an unstable variant with decreased oxygen affinity. Characterization of its synthesis, functional properties, and structure

In Hb Warsaw Val replaces the Phe normally present at the heme contact position beta 42 (CD1). This variant is unstable, and it readily undergoes methemoglobin formation. In DEAE-cellulose chromatography, the variant hemoglobin co-eluted with Hb A; a partially heme-depleted fraction of the variant, representing 5-6% of the total hemoglobin, eluted separately and in pure form. The heme replete form of Hb Warsaw exhibited decreased oxygen affinity with a normal Bohr effect and normal cooperativity and interaction with 2,3-diphosphoglycerate (DPG). The heme-depleted Hb Warsaw had a higher oxygen affinity than that of Hb A, decreased cooperativity and 2,3-DPG interaction, and a very low alkaline Bohr effect. Gel filtration of the heme-depleted form showed it to exist entirely as alpha beta dimers. Globin chain synthesis by Hb Warsaw-containing reticulocytes followed a balanced alpha/beta ratio. In short-term synthesis experiments, a major portion of incorporated radiolabeled L-leucine was recovered from the dimeric, heme-depleted Hb Warsaw fraction, suggesting that subunit association precedes the incorporation of heme into the beta subunits in the post-synthetic assembly of this hemoglobin. Structural analysis of deoxyhemoglobin containing roughly equal proportions of normal and variant beta chains showed that the replacement leaves a cavity next to the heme that is large enough to hold a water molecule, which may account for the instability of Hb Warsaw. The heme and the pyrrol nearest to ValCD1 tilt into the cavity. The resulting increase in the tilt of the proximal histidine relative to the heme plane, coupled with a possible stretching of the Fe-N epsilon bond may account for the low oxygen affinity.