Hb Tetramer Research Articles

Effect of chronic cold and submergence on blood oxygen transport in the turtle, chrysemys picta

Whole blood oxygen equilibrium curves (O 2EC's) and related hematologic properties are reported for the turtle Chrysemys picta exposed to two experimental conditions. Summer turtles were maintained at 24°C with free access to air; winter turtles were submerged for 4–12 wk in N 2-bubbled water at 3°C. Half-saturation P O 2 's at 3°C for blood from summer and winter animals were 4.1 and 4.5 Torr, respectively. At 24°C, summerand winter P 50's were 20.2 and 22.7 Torr, respectively. The winter turtle P 50 values were lower than predicted since prolonged submergence effected a severe metabolic acidos; blood pH's for winter turtles were 0.65 pH unit lower than for summer animals at both temperatures. Cold submergence also had a profound influence on O 2EC shape. Winter turtle curves exhibited high O 2 affinity below P 50 while they were distinctly right-shifted above 50% S. Winter animals also exhibited reduced CO 2-Bohr coefficients (Δlog P O 2 /ΔpH) at 3 and 24°C. Prolonged submergence did not affect the animal's isohemoglobin profile (demontrated by isoelectric focusing) or [metHb]. The [ATP] and [DPG] in winter turtle red cells, however, decreased significantly; the ratio of organic phosphate ([ATP]+[DPG]) to Hb tetramer fell from 1.4 in summer animals to 0.5 in winter turtles. These findings suggest that the effect of chronic cold and prolonged submergence on turtle O 2EC position and shape may result from reduction in RBC organic phosphates. Furthermore, these observed changes in blood oxygen transport may facilitate O 2 loading during winter submergence via extrapulmonary gas exchange.

Kinetic Studies on the Reconstitution of Deoxyhemoglobin from Isolated α and β Chains

The reconstitution reaction of deoxy hemoglobin (Hb) tetramer from isolated alpha and beta chains was kinetically studied by measuring circular dichroism (CD) changes in the Soret and the ultraviolet regions and optical absorbance change in the Soret region with a stopped-flow apparatus. The CD change in the Soret region was a fast reaction, followed by that in the ultraviolet region and the absorbance change in the Soret region. This fast reaction followed a simple second-order rate law with a rate constant of 6.4 x 10(5) M-1 . s-1. These results indicated that the combination of alpha and beta monomers into an alpha beta dimer accompanied the CD change in the Soret region and was the rate-limiting step of the overall reconstitution reaction of deoxyHb tetramer. On the other hand, the CD change in the ultraviolet region was ascribed to the combination of two alpha beta dimers into an Hb tetramer. The absorbance change in the Soret region was related to both the combination of alpha and beta monomers and that of two alpha beta dimers. From the analyses of these reactions the rate constant of the combination of two alpha beta dimers was determined to be 1.0 x 10(6) M-1 . s-1.

Neutron diffraction reveals oxygen-histidine hydrogen bond in oxymyoglobin.

Myoglobin (Mb) reversibly binds molecular oxygen in vertebrate muscle and consists of a polypeptide chain of 153 residues and one haem, which closely resembles one subunit of a haemoglobin (Hb) tetramer. In oxygenated myglobin (oxyMb) the iron atom is coordinated by four porphyrin nitrogen atoms, Ne of the invariant ‘proximal’ histidine (F8), and molecular oxygen1. The oxygen molecule lies in a tight pocket, bounded by two hydrophobic groups (Phe CD1 Val E11) and the side chain of the ‘distal’ histidine (E7). This histidine is present in Hb and Mb of many different organisms, with substitution by glutamine or leucine found in only a few cases. The function of the residue is not clear, although it does present steric hindrance to linear ligands such as carbon monoxide and favours ‘bent’ ones, such as O2. We report here that the imidazole stabilizes bound molecular oxygen with a hydrogen bond, as revealed by neutron diffraction analysis.

Hemoglobin Crete (beta 129 ala leads to pro): a new high-affinity variant interacting with beta o -and delta beta o -thalassemia

Hemoglobin Crete, beta129 (h7)ala leads to pro, is a new mutant hemoglobin (Hb) with high oxygen affinity that was discovered in a Greek family in various combinations with beta- and deltabeta- thalassemia. The propositus, who presented an unusual clinical picture of an “overcompensated” hemolytic state, with erythrocytosis, splenomegaly, abnormal red cell morphology, and marked erythroid hyperplasia, appeared doubly heterozygous for Hb Crete and deltabeta- thalassemia. His red cells contained 67% Hb Crete and 30% Hb F, and the combination of these two hemoglobins resulted in a blood P50O2 of 11.2 mm Hg. A brother with Hb Crete trait (38% Hb Crete, 56% Hb A, blood P50O2 23.0 mm Hg) did not have significant erythrocytosis. Purified Hb Crete was heat-unstable and exhibited a high oxygen affinity, and a normal Bohr effect. We postulate that the beta 129 proline substitution disrupts the H helix, perturbing nearby residues involved in alpha 1 beta 1 contact sites of the Hb tetramer.

Hemoglobin Crete (0129 Ala → Pro): A New High-Affinity Variant Interacting With β°-and β°-Thalassemia

AbstractHemoglobin Crete, β129 (H7) ala —► pro, is a new mutant bemoglobin (Hb) with high oxygen affinity that was discovered in a Greek family in various combinations with β° and δβ°-thalassemia. The propositus, who presented an unusual clinical picture of an “overcompensated” hemolytic state, with erythrocytosis, splenomegaly, abnormal red cell morphology, and marked erythroid hyperplasia, appeared doubly heterozygous for Hb Crete and δβ°-thalassemia. His red cells contained 67% Hb Crete and 30% Hb F, and the combination of these two hemoglobins resulted in a blood P50o2 of 11.2 mm Hg.A brother with Hb Crete trait (38% Hb Crete, 56% Hb A, blood P50o2 23.0 mm Hg) did not have significant erythrocytosis. Purified Hb Crete was heat-unstable and exhibited a high oxygen affinity, moderately decreased cooperativity, and a normal Bohr effect. We postulate that the 0129 proline substitution disrupts the H helix, perturbing nearby residues involved in α1β1, contact sites of the Hb tetramer.

An estimation of the first binding constant of O2 to human hemoglobin A.

Optical difference spectrophotometry has been applied for measuring the early has been applied for measuting the early part of the O2 dissociation curve of hukman hemoglubin (HP); i.e., below 1% O2 saturation levels or log [y(1–y)] form -3.0 to -2.0, where y=fractional O2 saturation. Two matched cuvettes, sealed on glass tonometers of known volume, were filled with the same deoxygenated Hb solution (65 μM in tetramer) under atmospheric pressure of pure argon. The pO2 values were calculated after the addintion to the sample tonometer of accutate volumes of O2/argon mixtures the composition of which was varied by using a precise gas-micing pump. 4–5 points of O2 saturation below y=0.01 were thus measutred, which allowed for the calculatiojn of A1, the first association constant of O2 to the tetrametric Hb. We have compared the effects pf protons, chloride ion s, CO2 and bisphosphoglycerate (P2-glycerate) on the pH dependence (alkaline Bohr effect(of A1 and p50. At low chloride condcentration (0.005 M) the alkaline Bohr effect at A1 was identical to that measured at p50, indicatintg that exctly one-fourth of the Bohr protons were released at the first oxygenation step. A higher chloride concentration (0.15 M) A1 was decr e a sed and p50 was increased as was increased as was the Bohr effect/. CO2 addintion (pCO2 38 Torr, 5050 Pa, at I=0.15 M) lead to decreased A1 and increased p50 but to a large decrease of the alkjaline Bohr effect. The effect of CO2 was predominat at A1, indicating a large releage of carbamino adducts from the tetrameric Hb at the first step fo ligation. P2-glycerate decreased A1 adn increases p50 values. The alkaline Bohr effect was enhanced at p50 by P2-glycerate but lowered at A1. These results are compared to those already published by other workers. The vartiations of the pH dependence of A1 in the presence of effectors indicate that A1 should be consifdered as a phenomenological parameter refering to one of the posssible structures that the Hb tetramer can take in the unliganded state. They indicate also that the whole Hb molecule feels the first oxygenation step, which itsilf implies structural changes within the quanternary structure.

Protein-protein interactions: Possible location of hemoglobin-haptoglobin contacts

We have studied the binding of α and β chain tryptic peptides to Hp linked to agarose. The results obtained provide evidence for a specific binding of peptides to Hp. Addition of Hb, which binds irreversibly Hp resulted in a specific displacement of the peptides bound to Hp. The analysis of the peptides displaced and their location in the Hb molecule suggested that Hb interacts with Hp through surfaces by which (I) αβ dimers and (II) α and β monomers face each other in the Hb tetramer.

Is the trimodality of Hb Leslie (alpha 2 beta 2 131 Gln---O) in heterozygotes the result of a variable number of active alpha-chain genes? Evidence for posttranslational control of hemoglobin synthesis.

Whether the trimodality in the relative concentration of the hemoglobin variant Hb Leslie in heterozygotes (Huisman, Hemoglobin 1:349-382, 1977) is due to a polymorphism of the alpha-chain structural genes was investigated by conventional incubation of reticulocytes with 14C-leucine. In addition, an aliquot from each of the incubations was incubated under the same conditions but without isotope. Three Hb Leslie heterozygotes with presumably four, three (heterozygous alpha-thalassemia-2), and two (homozygous alpha-thalassemia-2) active alpha-chain genes and with 33%, 22% and 11% Hb Leslie respectively, and one patient with the Hb Leslie beta(0)-thalassemia condition with more than 85% Hb Leslie were studied. The data indicate that betaLeslie chains have a lower affinity for alpha chains that betaA chains. A concomitant alpha-chain deficiency results in a reduced incorporation of betaLeslie chains into the tetrameric Hb Leslie molecules, while the quantity of Hb Leslie produced correlates with the degree of alpha-chain deficiency. Excess of betaLeslie chains is preferentially degraded.

Mechanism of inhibition of hemoglobin S polymerization by cyanate.

We have studied the effect of carbamylation on hemoglobin S (HbS) polymerization with the use of a new quantitative gelling technique. HbS gels fromed in the presence of nitrogen and dithionite (with or without carbon monoxide) at pH 7 in 0.15M phosphate were separated into sol and gel phases by centrifugation at 130,000 g. Hb concentration to the sol phase ([Hb]sol) of nonligated HbS was found to be constant (10.5 +/- 092 mM heme) over a range of original Hb concentrations from 11 to 17 mM at 24degrees C. This suggests that the HbS sol-gel equilibrium behaves as simple two-phase system. Increasing levels of total carbamylation from 0.65 to 3.65 moles CNO-/mole Hb tetramer (0.36 to 2.2 moles N-terminal/mole Hb4) progressively increases [Hb]sol. Specific activity of 14CNO-HbS was similar in the sol and gel phases, whereas COHbS appeared to be completely excluded from polymer structure of the gel. A comparison of the solubility of uncarbamylated and heavily carbamylated HbS at Co saturations ranging from 3 to 61 percent showed that the larges difference in [Hb]sol occured at the lowest ligand saturation rather than at intermediate states of ligation. Inhibition of HbS polymerization by carbamylation under these conditions, therefore, is not primarily the result of an effect on the T in equilibrium R comformational equilibrium. Our findings indicate that cyanate can interfere with HbS polymerization directly, possibly by alteration of surface binding sit(s) which are critical to aggregation. This direct action of cyanate may contribute significantly to the hematologic improvement achieved by cyanate treatment in sickle cell disease.

Relations between optical spectrum and structure in nitrosyl hemoglobin and hybrids.

The addition of inositolhexaphosphate to stripped nitrosyl hemoglobin provokes strong changes in the visible absorption spectrum of the protein. The stoichiometry of this reaction, determined by spectrophotometry, is one mole of IHP per tetramer. The equilibrium dissociation constant of IHP for Hb NO , in 0·1 m -2,2-bis (hydroxymethyl)-2,2′,2″-nitrilotriethanol (pH 6·8) is 1·3×10 −6 m and increases with ionic strength and pH. IHP has only a small effect on bis ( N -maleimidomethyl) ether hemoglobin. The spectral changes induced by IHP on hemoglobin nitrosyl hybrids α 2 CO β 2 NO and α 2 NO β 2 CO have been determined. Only the latter exhibits large changes in absorbance. Comparison between the Δ∈ values found for Hb NO and α 2 NO β 2 CO suggests that in Hb NO , the α NO subunit alone contributes to the spectral changes. IHP modifies also the ultraviolet absorption spectrum of Hb NO , and decreases the reactivity of cysteine β93 toward p -chloromercurybenzoate. These changes are qualitatively similar to those that appear on deoxygenation of oxyhemoglobin, and on binding IHP to aquomethemoglobin, as previously documented by Perutz et al. (1974 a ) . The suggestion that IHP induces a new quaternary conformation in Hb NO with deoxy character is also supported by the finding that spectral changes exactly opposite to those described in the reaction of IHP with Hb NO are induced in the absence of IHP after fixation of a ligand to α 2 NO β 2 deoxy hybrid, a reaction that probably consists of a change in the structure of this hybrid from a deoxy to an oxy conformation. The results are discussed in terms of the stereochemical hypothesis suggested by Perutz et al. (1974 b ) , according to which the allosteric equilibrium constant L in hemoglobin is governed by the Fe−N 8His distance. Kinetic studies have shown that the optical changes in Hb NO and α 2 NO β 2 deoxy hybrid are slow and complex, involving Hb NO dimers and tetramers. First and indirect estimates of the tetramer-dimer equilibrium constant of Hb NO are given. They are K 4/2 =4·2×10 −6 m and 20×10 −6 m , respectively, in 0·1 m -phosphate and 0·05 m -2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol buffers (pH 6·8). It is also demonstrated that the IHP-induced spectral changes in Hb NO owe their origin to a slow rearrangement of the α NO subunit inside a quaternary structure which has already changed in conformation, as suggested by the criterion of cysteine β93 reactivity. Evidence is provided that artificially prepared α 2 NO β 2 deoxy hybrid differs some-what in conformation from naturally occurring hemoglobin intermediates, as was suggested already for valency hybrids by Cassoly & Gibson (1972) .

Characterization of Heinz bodies in unstable haemoglobin haemolytic anaemia.

THE unstable haemoglobins are a group of well-defined haemoglobin variants associated clinically with haemolytic anaemia of varying severity1,2. The nature of the amino-acid substitutions reduces the stability of the haemoglobin molecule, and this results in precipitation within the red cell and formation of characteristic inclusions called Heinz bodies. The instability of this group of haemoglobins is also shown by their precipitation from solution when subjected to mild heating. The cellular inclusion bodies are generally not present unless the patient has been splenectomized, but can usually be induced by incubating the blood at 37° C for up to 48 h. Although it is generally accepted that the Heinz bodies consist of denaturation products of the abnormal Hb, as yet they have not been analysed directly, and there is some speculation as to their actual constitution2–4. In particular, it has been postulated that only the abnormal chains of the tetrameric Hb molecule precipitate, and that these may be lacking haem groups4. Some work has been done on the inclusion bodies which occur in the thalassaemias, in which there is impaired production of one of the polypeptide chains. Fessas et al.5 have shown that in β-thalassaemia, the peptide map of the inclusion bodies closely resembles that of pure α chains, and Rachmilewitz et al.6 have shown that in Hb H disease (α-thalassaemia) the inclusions are primarily β-chains largely present as haemichromes.

Characterization of a previously unidentified hemoglobin fraction

The hemoglobin peaks designated as “unnamed”, Hb 1a+b, Hb A 1c, Hb A GSSG, Hb A 0, and Hb A 2 were separated from human red cell hemolysate by CM-Sephadex chromatography. The first peak of Hb, unnamed, was found to have 1 mole of pyridoxal phosphate per mole of Hb tetramer. Treatment of hemolysate with pyridoxal phosphate increased the total amount of Hb in this peak without changing the ratio of pyridoxal phosphate to Hb. Substantial amounts of pyridoxal phosphate were not found in any other Hb fraction. Cord blood hemolysate, consisting mainly of Hb F is pyridoxylated to a much smaller extent than adult hemoglobin.

Use of a Fluorescent Analogue of 2,3-Diphosphoglycerate as a Probe of Human Hemoglobin Conformation during Carbon Monoxide Binding

Abstract The interaction of human hemoglobin (Hb) with 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT) was studied at pH 6.0 and T/2 = 0.04. Binding of HPT was determined by quenching of its fluorescence upon interaction with Hb. HPT binds to deoxy-Hb with a stoichiometry of 1 mole per mole of Hb tetramer and with a higher affinity for the deoxy than the liganded form analogous to the binding of the physiologically important 2,3-diphosphoglycerate (DPG). Binding is prevented by either DPG or inositol hexaphosphate. A qualitative comparison of the time courses of HPT release and CO combination during the reaction of CO with deoxy-Hb indicates a marked lag in the release of HPT compared with CO binding. This led to the conclusion that during the course of CO binding, significant amounts of partially liganded Hb intermediates are formed and the release of HPT from the intermediates does not occur in proportion to the amount of CO bound. Quantitative analysis of the data, in the form of least squares fitting procedures, was carried out to examine the ability of some elementary models to describe the results. The best fit was obtained on the assumption that HPT is released after 3 CO molecules have bound to hemoglobin.

On the mechanism of hemoglobin-haptoglobin complex formation.

In order to elucidate the mechanism of complex formation between human haptoglobin, Hp II, and bovine hemoglobin, Hb, measurements of the peroxidase activity and histidine determinations, as well as spectrophtometric and optical rotatroy dispersion titration experiments were performed.Following the raaction by slowly mixing the constituents of the complex in small portinos, the geometry of the complex turns out to depend on the sequence of mixing of the reactants. Adding Hb to Hp, a final complex Hb2→ Hp is formed. Teh inverse sequence of mixing leads to a complex of identical composition (Hp → Hb2), but different arrangement of the constituents, as shown by differences in the accessibility of histidine residues in both types of the complex. As suggested by the comparison of the histidine balance of both complexes, Hb2→ Hp may be described by the combination of αβ‐dimers of Hb with Hp (Hp (αβ)4), while Hp → Hb2 corresponds to the complex with two Hb tetramers (Hp(α2β2)2).Investigating the saturation process of complex formation in greater detail by spectrophtometric titration and optical rotatory dispersion, intermediary complexes are observed which prove the mechanism of complex formation to be a consecutive association process.Both series of complexes, Hp → Hb and Hb → Hp, seem to generate closely related configurations characterized by similar spectral and functional properties.

Spectroscopic properties of the hemes of human adult hemoglobin and its subunits

The circular dichroism (CD) and difference spectra of human adult hemoglobin (Hb A) and its subunits in the deoxy-, oxy- and CO-forms are presented. All four absorption bands of the hemes were optically active in the hemoglobins. The positions of the CD bands observed roughly agreed with those of the corresponding absorption bands. Generally, the α-subunit had more positive ellipticity than the β-subunit, and the CD spectrum of Hb A could be roughly composed by taking the arithmetic mean of those of the subunits. Similarly, the difference spectra of the subunits, between oxy- or CO- and deoxy-forms, differed from each other. These facts suggest that the local milieu around the hemes was not the same in the subunits. Human fetal hemoglobin showed the same CD spectrum as Hb A. In the visible region, two additional bands around 520 and 580 mμ were observed in the CD spectra, but these bands were indiscernible in the absorption spectra. A positive CD band around 580 mμ of the deoxy-form appeared in the α-subunit and Hb A but not in the β-subunit. On the basis of the difference spectra, the difference in the absorption of the deoxy-hemes in the separated subunits and in the tetrameric Hb A was confirmed. In the ultraviolet region, the CD peak positions of a band around 310 mμ differed markedly from each other in the subunits, while those of a band around 260 mμ did not. The CD band around 260 mμ of the deoxy-forms was probably degenerated. A negative CD band around 280 mμ was assigned to the aromatic amino acid residues of the protein moiety, and the band was indiscernible in the α-subunit.

Formation and properties of hemoglobin β2Aβ2S

1. 1.|Hemoglobin β 2 A β 2 S was isolated by electrophoresis on starch granules from a mixture of β A-PHMB and β S-PHMB chains (sulfhydryl groups blocked with p-hydroxymercuribenzoate (PHMB)) which had been treated with cysteine to regenerate sulfhydryl groups. Evidence for the structure β 2 A β 2 S included an electrophoretic migration midway between β A and β S on starch gels, an s 20, w of 4.5 S, recovery of Hb A and Hb S after recombination with α chains, and recovery of β A-PHMB and β S-PHMB chains (identified by position on starch-gel electrophoresis) after treatment with PHMB. With available information it cannot be explained with certainty why Hb β 2 A β 2 S was recovered in the above manner but was not seen during hybridization of hemoglobins H and S at pH extremes. 2. 2.|Hemoglobin β 2 A β 2 S was stable: it did not dissociate spontaneously into β 4 A and β 4 S. On preliminary determination its oxygen equilibrium was similar to that of Hb H, and it did not form a complex with haptoglobin 1-1. 3. 3.|Unlike deoxyhemoglobin S( α 2 β 2 S), deoxyhemoglobin β 2 A β 2 S had a solubility in concentrated phosphate buffer similar to that of Hb H and Hb A, and at a concentration of 37.4 g per 100 ml did not undergo gelation. Therefore, the presence of two β S chains in Hb tetramer was not a sufficient condition for the molecular aggregating properties seen with Hb S.