Increased Oxygen Affinity Research Articles

Homoglobin Rouen ( α-140 (HC2) Tyr→His): alteration of the α-chain C-terminal region and moderate increase in oxygen affinity

Hb Rouen ( α140 (HC2) Tyr→His) is a moderately high oxygen-affinity variant that was found in coincidence with polycythemia vera in a French patient. This hemoglobin provides an example of an alteration of the C-terminus of the α-chain, a region involved in the mechanisms of allosteric regulation. The increase in oxygen-affinity and decrease in cooperativity of this variant is much smaller than that resulting from the same substitution in the β-chain. This model provides additional evidence for the inequivalence between the α- and β-subunits.

Hemoglobin Dallas ( α97(G4)Asn→Lys):functional characterization of a high oxygen affinity natural mutant

Hemoglobin Dallas, an α-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity ( p 1 2 = 1 mmHg at pH 7.3 and 20°C), diminished cooperativity (0n, the Hill coefficient = 1.7 ) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20°C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be atttributed to a more ‘relaxed’ T-state.

Structure-function relationships in the low-affinity mutant haemoglobin aalborg (Gly74 (E18)β → Arg)

Haemoglobin Aalborg (Gly74 (E18)β → Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates: it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)β → Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2·8 Å resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)β protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)β forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18β on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.

Oxygen-binding properties of hemocyanin from the giant african snail Archachatina maginata: pH and calcium ion effects

1. 1. Spectral and elemental analyses of hemocyanin from Archachatina maginata show close similarities to those of molluscan hemocyanin. 2. 2. The oxygen affinity shows a normal Bohr effect with generally low levels of cooperativity. 3. 3. Removal of divalent cations increases oxygen affinity with loss in cooperativity. 4. 4. Divalent cations stabilise the integrity of whole molecules. 5. 5. Alkalinity promotes dissociation of whole molecules.

Microvascular oxygen transport: impact of a left-shifted dissociation curve.

The impact of an increased hemoglobin oxygen affinity (decreased P50) on oxygen transport was evaluated in capillaries of the retractor muscle under nonhypoxic (FIo2 = 0.30 and 0.21) and hypoxic (FIo2 = 0.10) conditions in hamsters with normal oxygen affinity [control; P50 = 26.1 +/- 1.0 (SD) mmHg, n = 12] and in hamsters with an increased oxygen affinity [treated; P50 = 16.2 +/- 1.6 (SD) mmHg, n = 7] induced by chronic short-term administration of sodium cyanate. Using in vivo video microscopy and computer-aided image analysis, we determined oxygen saturation (SO2) and associated hemodynamic parameters in both arteriolar (n = 30 control, 18 treated) and venular (n = 25 control, 17 treated) capillaries. In response to hypoxia, systemic arterial PO2 decreased to 29.6 +/- 6.0 (SD) mmHg in control animals and 24.7 +/- 3.8 (SD) mmHg in treated animals associated with abrupt decreases in systemic arterial blood pressure and increases in respiratory rate. The decrease in SO2 across the capillary network during nonhypoxic ventilation was 13.3% SO2 for control animals and 11.0% SO2 for treated animals. During hypoxic ventilation, the decrease in SO2 was 9.1% SO2 in control animals and 8.7% SO2 in treated animals. Hemodynamic parameters were not significantly different in the two groups during hypoxia. Estimated end-capillary PO2 was significantly lower in the treated animals. These data indicate that an increased oxygen affinity does not provide an obvious advantage for oxygen transport during hypoxia at the level of the capillary network in resting striated muscle; however, such an advantage might become apparent in the presence of an increased metabolic rate or a more severe hypoxic challenge.

Structure of the EF corner favors deamidation of asparaginyl residues in hemoglobin: the example of Hb La Roche-sur-Yon [ β81 (EF5) Leu → His

Some abnormal hemoglobins constitute models which allow one to evaluate the structural requirements for post-translational modifications in proteins, such as deamidation. Hb La Roche-sur-Yon [ β81 (EF5) Leu → His] is an unstable hemoglobin variant displaying a moderately increased oxygen affinity. About half of the abnormal hemoglobin, in addition to the substitution at position β81, carries a deamidation of the neighboring asparagine residue, β80(EF4). The histidine at position β81 cannot fit into the small hydrophobic pocket which normally accomodates the leucine residue. This structural change opens the heme pocket and modifies the general conformation of the EF segment, thus explaining the increase in oxygen affinity and the achievement of a three-dimensional structure favoring asparagine deamidation. Histidine ß81 could also act as a catalyst in the deamidation reaction. Deamidation has already been reported for two other variants of the EF corner, Hb Providence [ ß82 (EF6) Lys → Asn] and Hb J Singapore [ α79 (EF8) Ala → Gly]. In all these cases it seems that a histidine may catalyze the deamidation of the asparagine residues and that disturbing the folding of the EF corner will provide an extra flexibility favoring the reaction.

Oxygen and Acid-Base Disturbances in the Hemolymph of the Lobster Homarus Gammarus During Commercial Transport and Storage

ABSTRACT European lobsters, Homarus gammarus, delivered to the wholesale market in Birmingham, England, had experienced 12-14 h of exposure to air during shipment from coastal suppliers. Lobsters judged to be in good condition on arrival at the market had low postbranchial oxygen partial pressures (PaO2, 1.9 kPa) but maintained circulating oxygen contents closer to submerged levels (CaO2, 0.27 mmol 1–1) chiefly by conserving oxygen combined to hemocyanin (CHc O2, 0.24 mmol 1–1). In addition, they had accumulated some CO2 (PaCO2, 1.13 kPa) and lactic acid (9.0 mmol 1–1) but were not acidotic (pHa, 7.71) due to an elevation in buffer base ([HCO3–]a, 21.7 mmol 1–1). These changes paralleled those observed in a laboratory study of exposure of H. gammarus to air, which revealed that internal buffering of a potential acidosis plus increased oxygen affinity of hemocyanin restored oxygen transport, enabling prolonged survival. On resubmergence, recovery of PaO2 and PaCO2 was complete within 1 h with a slower recovery in [HCO3–]a and an initial rise in lactate levels by 3.3 mmol 1–1 after 30 min, before a gradual return to submerged values over 24 h. Lobsters delivered in poor condition were severely hypoxemic (CaO2 and CHc O2 <0.1 mmol 1–1 ') due either to a pronounced and uncompensated acidosis (pHa, 7.21–7.57) or, in late premolt lobsters, to low oxygen-carrying capacities. On resubmergence, these lobsters showed abnormal patterns of oxygen and acid-base adjustments with only 25% of the animals sampled surviving 24 h in the holding tanks. The ability of lobsters to survive exposure to air during commercial shipment was influenced by the presence of a period of ponding before transport to market, journey time from supplier to market, transportation temperature, levels of disturbance, and molt stage.

Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering.

Of two closely related species of geese, one, the greylag goose, lives in the Indian plains all year round, while the other, the bar-headed goose, lives at the Tibetan lakes and migrates across the Himalayas to winter in India. Another species, the Andean goose, lives in the High Andes all year round. Possession of a Hb with high oxygen affinity helps to adapt bar-headed and Andean geese to high altitudes. The Hb amino acid sequences of the bar-headed and the greylag geese differ by four substitutions, of which only one is unique among bird sequences: Pro-119 alpha (H2)----Ala. Perutz proposed that the two-carbon gap left by this substitution at the alpha 1 beta 1 contact raises the oxygen affinity, because it relaxes the tension in the deoxy or T structure [Perutz, M. F. (1983) Mol. Biol. Evol. 1, 1-28]. It was later found that the Hb of the Andean goose has a gap in the same position, due to the complementary substitution Leu-55 beta (D6)----Ser. We have tested Perutz's hypothesis by introducing each of these substitutions into human globin synthesized in Escherichia coli. The reconstituted Hbs combine cooperatively with oxygen. Their oxygen affinities exceed that of normal human Hb by an even larger factor than that found between the high-flying geese and the greylag goose. The mutant Hb Met-55 beta (D6)----Ser was crystallized. Its structure is the same as that of HbA, except in the immediate environment of the gap left by the substitution of the serine for the methionine side chain, which evidently causes the increased oxygen affinity of this Hb.

Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin.

Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of approximately 70%. The predominant product was crosslinked between subunits within a tetramer and had a Mr of 64,000; no higher Mr species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.

Site-directed mutagenesis in haemoglobin: Functional role of tyrosine-42(C7)α at the α1-β2 interface

To clarify the functional role of Tyr-42(C7)α, which forms a hydrogen bond with Asp-99(G1)β at the α1-β2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42α was replaced by Phe (Hb Phe-42α) or His (Hb His-42α), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99β mutants, Hb Phe-42α showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42α, the deoxy T quaternary structure is highly destabilized and the strain imposed on the FeN ε (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42α, Hb His-42α showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42α and Asp-99β. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42α and Asp-99β plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.

Analysis of Oxygen Equilibria of the Giant Hemoglobin from the Earthworm &lt;italic&gt;Eisenia foetida&lt;/italic&gt; Using the Adair Model

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.

HB Cleveland or α2β293(F9)CYS→ARG;121(GH4)GLU→GLN

Hb Cleveland is characterized by two amino acid substitutions, namely beta 121(GH4)Glu----Gln as in Hb D-Los Angeles and beta 93(F9)Cys----Arg as in Hb Okazaki, and shares with Hb Okazaki a decreased stability, an increase in oxygen affinity, and decreases in Bohr effect and heme-heme interaction. It is the 13th beta chain variant with two substitutions that has been described thus far.

A Case of the Hb Regina (β 96 (FG3) Leu → Val) in a German Male Associated with High Oxygen Affinity and Erythrocytosis

Hb Regina was identified in a 58-year-old German male and 2 of his 3 children. All affected subjects presented moderate erythrocytosis and the whole blood exhibited increased oxygen affinity (P50:17.5 mm Hg). This hemoglobinopathy was undetectable with the conventional electrophoretic methods. It was, however, separated and quantified by cation-exchange and reverse-phase high-performance liquid chromatography. Hb Regina accounted for 30-35% of the total Hb. No significant clinical symptoms were found to be related to this hemoglobinopathy. This is the first known instance in Germany, and so far the second case reported.

Changes of polymerization and conformation of hemoglobin S induced by thiol reagents

Thiol reagents, covalently bound to cysteine β93, either inhibit or facilitate the polymerization process of hemoglobin S. The progelling effect of parahydroxymercurybenzoate or 2,2′-dithiodipyridine contrasted with the increased oxygen affinity and the destabilization of the T state of Hb shown by functional and NMR studies. Thiol reagents increased the oxygen affinity of Hb from 30 to 1000%. Such variability was also observed in the reduction (up to 50%) of the alkaline Bohr effect. We show that the antigelling or progelling activity of thiol reagents does not depend solely on the concentration of molecules present in the deoxy T state but that specific effects of the reagent affects molecular interactions of the hemoglobin S polymerization process.

Treatment of Sickle Cell Anemia with Hydroxyurea and Erythropoietin

Hydroxyurea increases the production of fetal hemoglobin (hemoglobin F) in patients with sickle cell anemia and therefore has the potential for alleviating both the hemolytic and vaso-occlusive manifestations of the disease. There is preliminary evidence that recombinant human erythropoietin may also increase hemoglobin F production. We treated five patients with sickle cell disease with escalating doses of intravenous erythropoietin for eight weeks. Three of these patients were subsequently treated with daily doses of oral hydroxyurea. After the optimal dose was determined, erythropoietin was then given along with hydroxyurea for four weeks. Treatment with erythropoietin, either alone or in combination with hydroxyurea, had no significant effect on the percentage of hemoglobin F-containing reticulocytes (F reticulocytes) or red cells (F cells). In contrast, hydroxyurea treatment was associated with a 3-to-25-fold increase in F reticulocytes, a 1.6-to-7-fold increase in F cells, and a 2.3-to-16-fold increase in the percentage of hemoglobin F. In all three patients given hydroxyurea, treatment with this drug was associated with reduced hemolysis, shown by decreases in serum bilirubin and lactic dehydrogenase and prolongation of red-cell survival. Hydroxyurea treatment also resulted in a decrease in the percentage of irreversibly sickled cells and sickling at partial oxygen saturation, an increase in oxygen affinity and total red-cell cation content, and a reduction in potassium-chloride cotransport. All three patients had a decrease in the number of pain crises. This study confirms that hydroxyurea therapy increases hemoglobin F production and provides objective evidence that hydroxyurea reduces the rate of hemolysis and intracellular polymerization of hemoglobin S. In contrast, recombinant human erythropoietin, whether alone or in combination with hydroxyurea, offers no measurable benefit.

Oxygen binding to partially oxidized hemoglobin

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.

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the .beta. 27 (B9) position

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.

A New α Chain Variant HB Tonosho [α110(G17)ALA→THR]: Subunit Dissociation During Cation Exchange Chromatography for HB A1cAssay

A new alpha chain variant, alpha 110(G17)Ala----Thr, was detected because of subunit dissociation during the determination of the Hb A1c by automated cation exchange high performance liquid chromatography. The abnormal hemoglobin overlapped the cathodic edge of the band of Hb A in isoelectrofocusing. It was slightly unstable in the isopropanol test and had a slightly increased oxygen affinity. The abnormal alpha chain eluted slightly faster than the normal alpha chain in reversed phase high performance liquid chromatography. The amino acid substitution was determined by purification of S-alkylated alpha T-12,13 tryptic peptide, chymotryptic digestion, and sequencing of an octapeptide alpha 110-117. The abnormal alpha chain comprised about 14% of the total alpha chain. A biosynthetic study did not suggest selective loss of the abnormal chain in reticulocytes.

HB Mizuho [β68(E12)LEU-PRO]. Second Occurrence Identified in A Caucasian Child with Hemolytic Anenia and Dense Erythrocyte Inclusions

Hb Mizuho [beta 68(E12)Leu----Pro] was identified in a child of Italian/Sicilian descent who exhibited severe, transfusion dependent hemolytic anemia which improved following splenectomy. The patient's peripheral blood smear, which prior to splenectomy demonstrated coarse erythrocytic basophilic stippling, showed large, dense erythrocytic hemoglobin inclusions following splenectomy. Whole blood oxygen equilibrium results were consistent with the presence of a hemoglobin component exhibiting increased oxygen affinity with decreased cooperativity. The abnormal beta chain was characterized by high performance liquid chromatography analysis of the isopropanol precipitable hemoglobin fraction.

Control and coordination of gas transfer in fishes

Recent developments pertaining to the control and coordination of gas transfer in fishes have been reviewed. Gill ventilatory water flow can markedly affect blood respiratory and blood acid–base status. Although arterial oxygen content traditionally has been considered the predominant factor controlling ventilation, we present evidence for additional involvement of both blood acid–base status and circulating catecholamines. An analysis of the independent effects of blood oxygen content, acid–base status, and catecholamines in controlling ventilation is confounded by the interrelationships among these variables. It is likely, however, that each factor is involved to some extent in ventilatory control in fishes. Blood oxygen transport is affected by the carrying capacity of the blood and red blood cell chemical status. Blood oxygen-carrying capacity is increased during periods of stress by adrenergic release of red blood cells from the spleen. Concurrently, adrenergic stimulation of red blood cell Na+–H+ exchange, reduction of intracellular nucleoside triphosphates, swelling of red blood cells, and respiratory alkalosis all tend to increase oxygen affinity and capacity of hemoglobin. Results of recent in vivo studies indicate that adrenergic inhibition of plasma bicarbonate dehydration may contribute to the respiratory acidosis after exhaustive exercise in fishes. Evidence is presented to show that hypoxemia, rather than blood acidosis per se, is the proximate stimulus for catecholamine mobilization during periods of stress in fishes.