Effect of organic co-solvents on the dimer/tetramer equilibrium of human haemoglobin

We have studied the effect of organic co-solvents (monohydric alcohols and formamide) on the dimer-tetramer equilibrium of human haemoglobin by measuring the dependence of oxygen affinity upon haemoglobin concentration over a 100-fold concentration range and analysing the data with a modified Monod-Wyman-Changeux model in which the equilibrium (tetramer) in equilibrium with (dimer) + (dimer) was taken into account. This procedure enabled us to obtain the dimer-tetramer equilibrium constant and to find its dependence upon co-solvent concentration. Then by following the procedure already reported for the tense----relaxed state transition of haemoglobin, we separated the co-solvent effects into bulk electrostatic and non-bulk electrostatic (hydrophobic) contributions. We believe that our results demonstrate that during haemoglobin dissociation, just as we have already shown for the tense to relaxed state transition, both charged groups and hydrophobic surfaces became exposed to the solvent.     

Oxygen affinity of bovine haemoglobin. Relevance of electrostatic and hydrophobic interactions

We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen equilibrium of bovine haemoglobin and have compared them with the effects of the same cosolvents on the oxygen equilibrium of human haemoglobin. Our results indicate: (1) that in agreement with previous suggestions, the lower affinity of bovine haemoglobin for oxygen is not due to an increased number of salt bridges stabilizing the T structure; (2) that, following T----R transition, more hydrophobic surface is exposed to the solvent by bovine than by human haemoglobin. We suggest, therefore, that a relevant role in keeping the oxygen affinity of bovine haemoglobin lower than that of human haemoglobin is played by the higher free energy needed to expose this more hydrophobic surface to the solvent. We stress, however, that our analysis does not enable us to say which particular amino acid residues are concerned in these effects.     

Ligand binding kinetics of des arginine haemoglobin

Des arginine 141 a haemoglobin (the haemoglobin in which the C-terminal arginine of the a chain has been removed) has a high affinity for oxygen and a reduced co-operativity in its oxygen equilibrium binding. The kinetic consequences of this modification are investigated in this paper. Deoxy des Arg haemoglobin binds carbon monoxide faster than does haemoglobin A, whilst oxy des Arg haemoglobin loses oxygen more slowly. These results are correlated with the oxygen equilibrium binding properties of des Arg haemoglobin. The carbon monoxide binding kinetics have been interpreted as implying a change in the parameter c (of the allosteric model), as well as L, when this arginine is removed from haemoglobin.     

Haemoglobins of invertebrate tissues. Nerve haemoglobins of Aphrodite, Aplysia and Halosydna

1. The occurrence of haemoglobin in invertebrate nerves is surveyed. Haemoglobin was observed in the nerves and ganglia of the marine nematode Amphiporus sp. and of the polychaet annelid Halosydna sp. 2. Haemoglobins from the nerve and ganglia of the polychaet annelid Aphrodite aculeata L. and from the nerve of the gastropod mollusc Aplysia californica have been partially purified. The haem in each case was identified as iron protoporphyrin IX. 3. The minimum molecular weight of Aphrodite nerve haemoglobin deduced from the haem content and amino acid analysis is 17090, in agreement with the molecular weight 15600+/-1000 determined by sedimentation equilibrium. 4. The molecular weight of Aplysia nerve haemoglobin was determined by sedimentation equilibrium to be 16400+/-1000. 5. The oxygen dissociation curves are hyperbolic. Half-saturation is achieved at 1.1mm. Hg for Aphrodite nerve haemoglobin and at 4.0mm. Hg for Aplysia nerve haemoglobin. The coefficients for partition between carbon monoxide and oxygen are: Aphrodite nerve haemoglobin, 167; Aplysia nerve haemoglobin, 116. 6. The ferrous haemoglobins combine with cyanide. 7. We conclude that the intracellular haemoglobins of muscle and nerve are similar.     

Studies on carbon monoxide binding by shark haemoglobin.

The kinetics of the reactions of Pacific-porbeagle haemoglobin with CO were studied by flash-photolysis and stopped-flow methods, and the equilibrium binding curves for CO were measured in spectrophotometric titrations. Measurements were made in the pH range 6-8 and in the temperature range 0-40 degrees C. The results are discussed in terms of the allosteric model proposed by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88-118]. Within this framework the results indicate that in the R-state the haem groups fall into two classes of different reactivity with different spectral characteristics, but that in the T-state the groups may be essentially equivalent. The physiological importance of the temperature-insensitivity of the equilibrium ligand-binding curves for porbeagle haemoglobin is discussed.     

Stereochemical mechanism of oxygen transport by haemoglobin

Spectroscopic and chemical evidence speak in favour of the iron-oxygen bond being polar. X-ray analysis shows that the oxygen molecule is inclined at an angle of about 115 degrees to the haem plane. Cooperative binding of oxygen by haemoglobin is due to an equilibrium between two alternative structures, which differ in oxygen affinity by the equivalent of 3-3.5 kcal/mol. I proposed that in the low affinity structure the globin opposes the movement of the iron atom from its five-coordinated pyramidal geometry in the haem of deoxyhaemoglobin to its six-coordinated planar geometry in the haem of oxyhaemoglobin, while in the high affinity structure this restraint is absent. Recent evidence supporting this mechanism is described.     

Modification of human haemoglobin with glucose 6-phosphate enhances tetramer-dimer subunit dissociation.

Studies using equilibrium gel-permeation chromatography demonstrate that formation of the covalent adduct of D-glucose 6-phosphate (G6P) with human haemoglobin promotes dissociation of the haemoglobin tetramer into its component alpha beta dimer pairs [Kdoxy = 2.57 X 10(-6) versus Kdoxy (G6P) = 11.22 X 10(-6) M-haem]. On the other hand, Kd for glucosylated haemoglobin is identical with those of the O2- and CO-liganded forms of intact haemoglobin A0. These data are consistent with the phosphate moiety alone being responsible for a 4.5-fold increase in the tetramer-to-dimer apparent Kd. This suggests the glucose 6-phosphate moiety does not bind to the same sites on haemoglobin as do the free organic phosphates, as suggested by ligand-binding kinetics data or structural analysis. My study presents a working model for studying changes in protein subunit assembly as altered by protein phosphorylations.     

Studies on the interaction of chlorpromazine with haemoglobin

The interaction of chlorpromazine (CPZ), a widely used antipsychotic tranquillizer, with the allosteric protein haemoglobin, has been studied by different methods. From r versus Cf plot obtained by an equilibrium dialysis experiment, the maximum value of r was found to be 6.8 at 0.15 M NaCl. Binding parameters, namely the affinity constant K and the degree of cooperativity nH, were determined from the Hill plot. Circular dichroism studies indicate a conformation change of haemoglobin in the presence of CPZ. Oxygen has been found to be released from haemoglobin with the progressive addition of CPZ. The extent of the release of oxygen depends on the stoichiometric ratio of CPZ: haemoglobin (D/P). The possible nature of the binding site of the protein has been discussed on the basis of the information obtained from fluorescence measurements.     

A mathematical model for the computation of carboxyhaemoglobin in human blood as a function of exposure time.

A mathematical model is developed for the carbon monoxide (CO) uptake by the blood by taking into account the molecular diffusion, convection, facilitated diffusion and the non-equilibrium kinetics of CO with haemoglobin. The overall rate for the combination of CO with haemoglobin is derived by including the dissociation of CO from carboxyhaemoglobin (COHb). The resulting coupled system of nonlinear partial differential equation with physiologically relevant initial, entrance and boundary conditions is solved numerically. A fixed point iterative technique is used to deal with nonlinearities. The concentration of COHb in the blood is computed as a function of exposure time and ambient CO concentration. The COHb levels computed from our model are in good agreement with those measured experimentally. Also, results computed from our model give better approximation to the experimental values compared with the results from other models. The time taken by the blood COHb to attain 95% of its equilibrium value is computed. The COHb concentration in the blood increases with the increase in ventilation rate, association rate coefficient of CO with haemoglobin and total haemoglobin content in the blood, and with the decrease in dissociation rate coefficient of CO with haemoglobin and mean capillary blood PO2. It is found that the COHb level in the blood is not affected significantly because of endogenous production of CO in the body under normal condition. However, the effect may be significant in the patients with haemolytic anaemia.     

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.     

Differential scanning microcalorimetry study of the thermal denaturation of haemoglobin

A study of thermal denaturation of human haemoglobin A0 (HbA0) and methaemoglobin (mHb) was carried out by differential scanning calorimetry. DSC haemoglobin profiles were scan rate dependent and only partly reversible. Thermal unfolding of protein was analysed with the use of both equilibrium thermodynamic and kinetic approaches. The fittings based on the simple equilibrium/dissociation model were good and much more satisfactory than those based on "fully-kinetic" models. However the presence of some kinetic distortion during the unfolding process should be noted due to the scan-rate effect on DSC transitions. The calculated first-order kinetic constant for mHb was higher by two orders than the one for HbA0 (stabilised form). The average activation energy for HbA0 was found to be 289 +/- 28 kJ M-1 while for mHb it was about 100 kj M-1 lower.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

The role of a 70 kDa nuclear protein in fetal haemoglobin synthesis in K562 cells

Abstract. Previously, we reported that a 70 kDa nuclear protein may regulate fetal haemoglobin gene expression in haemin treated K562 cells. To obtain further evidence of the specific role of this 70 kDa nuclear protein, we compared the nuclear fractions isolated from phenylacetate, hydroxyurea and haemin treated K562 cells. Both phenylacetate and hydroxyurea have been used to induce fetal haemoglobin synthesis in K562 cells. Cell growth was measured by biochemical events including DNA, RNA and protein synthesis. Differentiation of K562 cells was determined by both [³H]-leucine incorporation into fetal haemoglobin and scoring benzidine-stained positive cells. Unlike the haemin treated cells, phenylacetate and hydroxyurea induced growth arrest and increased fetal haemoglobin gene expression in K562 cells. After four days of treatment with phenylacetate and hydroxyurea more than 50% of the cells stained positive with benzidine. The SDS-Polyacrylamide gel electrophoretic analysis of nuclear proteins isolated from phenylacetate and hydroxyurea treated K562 cells showed that the 70 kDa protein was reduced in nuclear protein extract in both groups similar to haemin treated cells. These results suggest that the loss of the 70 kDa protein from a nuclear protein extract is not restricted to only haemin treated cells but also occurs in hydroxyurea and phenylacetate treated cells. Our results provide further evidence that the 70 kDa nuclear protein may be involved in regulating fetal haemoglobin expression through a negative control mechanism.     

Organotin-protein interactions. Binding of triethyltin bromide to cat haemoglobin.

Triethyltin binds to native cat and rat haemoglobin but not to their denatured forms or to other animal haemoglobins. Two molecules of the organotin bind to one molecule of R-state cat haemoglobin with affinity constants of about 1 X 10(5) M-1. Little or no triethyltin is bound to the deoxygenated (T-state) protein. Binding appears to be dependent upon the existence of a specific three-dimensional configuration of cysteine and histidine residues. The properties of the triethyltin-cat haemoglobin complex are consistent with those of a haemoglobin conformer whose allosteric equilibrium is displaced toward the R-state. Its oxygen affinity and rate of oxidation by nitrite is increased, and the rate of reduction of the methaemoglobin derivative by ascorbate is decreased. These effects of triethyltin are opposite and antagonistic to the effects of inositol hexaphosphate. They are exerted on the alpha- as well as beta-haem groups, even though triethyltin is bound at sites on alpha-globin far removed from the haem groups.     

Clinical Studies and Physiological Properties of Hopkins-2 Haemoglobin

HAEMOGLOBIN Hopkins-2 (Ho-2) was discovered in a family in which haemoglobin S was also present¹. Independent segregation of the two abnormal haemoglobins provided the first convincing evidence that two genetic loci are concerned with synthesis of the haemoglobin molecule^1,2. Subsequently, evidence was obtained that the abnormality in haemoglobin Ho-2 is in the alpha chain³, whereas the lesion in haemoglobin S is in the beta chain of globin. We have reported the structural abnormality in Ho-2 and we now present a revised pedigree and the clinical status of carriers; we attempt to relate these findings to functional abnormalities exhibited by the haemoglobin.     

Clinical Studies and Physiological Properties of Hopkins-2 Haemoglobin

HAEMOGLOBIN Hopkins-2 (Ho-2) was discovered in a family in which haemoglobin S was also present¹. Independent segregation of the two abnormal haemoglobins provided the first convincing evidence that two genetic loci are concerned with synthesis of the haemoglobin molecule^1,2. Subsequently, evidence was obtained that the abnormality in haemoglobin Ho-2 is in the alpha chain³, whereas the lesion in haemoglobin S is in the beta chain of globin. We have reported the structural abnormality in Ho-2 and we now present a revised pedigree and the clinical status of carriers; we attempt to relate these findings to functional abnormalities exhibited by the haemoglobin.     

Non-equilibrium state of a monomeric insect haemoglobin induced by γ-irradiation and detected by Mössbauer spectroscopy

The met-aquo form of the monomeric insect haemoglobin CTT III has been investigated by Mössbauer spectroscopy before and after reduction with thermolyzed electrons at low temperature. The native met haemoglobin dissolved in water and water/glycerol mixtures, respectively, exhibits in the range of pH 5.8 to 9.0 high-spin iron(III). The electronic state of the haemoglobin is not affected by the solvent conditions. In water/glycerol γ-irradiation at 77 K results in the reduction of the haem iron by thermolyzed electrons. Due to this process, the hexacoordinated high-spin iron(III) is transformed into a hexacoordinated low-spin iron(II). This latter complex is a transition state which changes into the high-spin iron(II) state of the deoxyhaemoglobin when increasing the temperature. Thus, a kinetically stabilized non-equilibrium state of the haemoglobin exists at low temperature which relaxes with increasing temperature and finally reaches the equilibrium state to form deoxyhaemoglobin. This transition occurs at T > 190 K and corresponds with drastic changes in the temperature dependence of the Lamb-Mössbauer factor. Both effects indicate an alteration of the intramolecular flexibility of the haemoglobin.     

States of stability/lysis in human fetal and adults red blood cells

In human red blood cells from umbilical cord (NRBC) and from adult (ARBC) blood we measured: (i) the equilibrium distribution (medium/cells) for haemoglobin as a function of medium osmolality (osmotic fragility curve) and (ii) the rate of haemoglobin loss to a hypotonic medium of fixed osmolality. From the analysis of the osmotic (cumulative) fragility curve, a subpopulation of high resistance (young?) cells was individualized only in samples from cord blood. The differences presented in the rate of haemoglobin loss between samples of umbilical cord and adult's blood point to distinct cell surface restrictions (cytoskeleton and/or plasma membrane phospholipid) to haemoglobin leak in both cells. Uniformly distributed (among all cells in both samples) differences could explain the distinct rates of haemoglobin loss. However, marked differences restricted to only a subpopulation of cord blood cells could also explain these results.     

Some factors affecting oxygen uptake by red blood cells in the pulmonary capillaries

In this study we investigate the equations governing the transport of oxygen in pulmonary capillaries. We use a mathematical model consisting of a red blood cell completely surrounded by plasma within a cylindrical pulmonary capillary. This model takes account of convection and diffusion of oxygen through plasma, diffusion of oxygen through the red blood cell, and the reaction between oxygen and haemoglobin molecules. The velocity field within the plasma is calculated by solving the slow flow equations. We investigate the effect on the solution of the governing equations of: (i) mixed-venous blood oxygen partial pressure (the initial conditions); (ii) alveolar gas oxygen partial pressure (the boundary conditions); (iii) neglecting the convection term; and (iv) assuming an instantaneous reaction between the oxygen and haemoglobin molecules. It is found that: (a) equilibrium is reached much more rapidly for high values of mixed-venous blood and alveolar gas oxygen partial pressure; (b) the convection term has a negligible effect on the time taken to reach a prescribed degree of equilibrium; and (c) an instantaneous reaction may be assumed. Explanations are given for each of these results.     

Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone.

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.