Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Physical, biochemical and functional characterization of haemoglobin from three strains of Artemia

The brine shrimp, Artemia, an inhabitant of coastal and inland salterns, encounter fluctuations in the salinity which in turn influences the oxygen availability of their habitat. Hence, experiments were performed to analyze variations in haemoglobin structure and patterns of three strains of Artemia from South India and also to reflect the effect of varying oxygen levels in their habitat. Haemoglobins were purified on a DEAE-Sephadex column and haemoglobin types were analyzed by comparing their relative mobility on a non-denaturing medium. Furthermore, their molecular masses were determined by gel filtration in Sepharose column and by dodecylsulfate polyacrylamide gel electrophoresis. Results clearly reveal the presence of three distinct extracellular haemoglobins Hb I, Hb II and Hb III in Tuticorin strain while the other strains displayed only trails or the complete absence of Hb III and Hb II. Estimated molecular masses of these haemoglobins are 235,000-250,000 Da. Denaturation of the reduced and alkylated haemoglobins revealed apparently one polypeptide chain with a molecular mass of 124,000 Da. Upon denaturing gel electrophoresis of native haemoglobin Hb II, it was found that the 124,000 Da, polypeptide was cleaved specifically into two unequally-sized fragments of 50,400 and 79,800 Da. With regard to oxygen affinity, Hb III has a very high affinity for oxygen, an almost negligible Bohr effect and a good physiological adaptation to temperature changes. By combining the three haemoglobins in different proportions Artemia strains must be able to withstand diverging environmental conditions. In particular, the absence of Hb III in Puthalam and its occurrence as a faint band in Thamaraikulam could be correlated to the oxygen levels of their habitats.     

Oxygen transport to tissue modified by entrapment of an allosteric effector of haemoglobin in erythrocytes

Oxygen affinity of haemoglobin is modulated by several parameters such as the allosteric effector 2-3 DPG for most mammalians. Inositol hexaphosphate (I.H.P.) exerts the same effect on haemoglobin. A previously developed new methodology for the entrapment of drugs into erythrocytes has been adapted to I.H.P.; it is based on a reversible osmotic shock. I.H.P. loaded red blood cells have characteristics very similar to those of native cells. The decrease in oxygen affinity is related to the dose of encapsulated I.H.P. In piglets, transfusion of such cells has led to an increase of oxygen extraction from haemoglobin. Normal anesthetized animals regulate their oxygen consumption by reduction of cardiac output.     

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.     

Familial Polycythaemia Caused by a New Haemoglobin Variant: Hb Heathrow, β 103 (G5) Phenylalanine Leucine

A new variant of haemoglobin A (Hb A) with a high affinity for oxygen has been found in an English family. Five members are affected and all are polycythaemic. This variant (Hb Heathrow) is the first of this class to be found in this country and has the same electrophoretic mobility as Hb A. It was discovered only by measuring the oxygen affinity of the patients'' red cells. This emphasizes the need for measuring the oxygen affinity of haemoglobin in patients with polycythaemia if other clinical and haematological features associated with polycythaemia rubra vera are absent.     

The effect of temperature on the ability of Tilapia mossambica Peters to enter deep water

The ability of adult Tilapia mossambica Peters to enter deep water was determined at 15, 22 and 30°C. At 30°C adults compensate to about 20m depth but at 15°C to only 7 m. Compensation is more rapid at high than at low temperatures. T. mossambica haemoglobin has a marked Root effect which is the same at 22 and 30°C. The oxygen affinity of the haemoglobin is higher at 15°C than at 30°C. There was no measurable difference in the rate of passive oxygen diffusion across the swimbladder wall in the temperature range 15–30°C. It is concluded that the ability to enter deeper water at higher temperatures is related to decreased oxygen affinity of the haemoglobin and higher rates of oxygen secretion and blood circulation.     

Studies on haemoglobin immobilized by cross-linking with glutaraldehyde. Cross-linked soluble polymers and artificial membranes

Human haemoglobin was immobilized by cross-linking with glutaraldehyde as soluble polymers and artificial membranes. Effects of pH and 2,3-diphosphoglycerate on oxygen binding and cross-linking were studied with haemoglobin immobilized in both the oxy and deoxy states. The cooperativity is suppressed and the affinity is increased when compared with native haemoglobin. Haemoglobin immobilized in the oxy state exhibited a higher oxygen affinity than that immobilized in the deoxy state. The alkaline Bohr effect is not significantly different from that of native haemoglobin. The 2,3-diphosphoglycerate influence on oxygen binding was reduced by one third with immobilization. In order to separate the chemical and the "conformation freezing' effects on the properties of immobilized haemoglobin, glutaraldehyde-modified haemoglobin in oxy and deoxy states was produced. Oxygen binding was studied and chemical modifications were checked by electrophoresis and gel filtration. This chemically modified haemoglobin without polymerization and without intra-chain bridging exhibits a behaviour similar to that of cross-linked soluble polymers or membranes of haemoglobin.     

Blood adaptations to marine and freshwater environments in fish of the Family Sciaenidae (Perciformes)

In five species of fish from the Family Sciaenidae, collected from marine, brackish and fresh-water environments, the following parameters were studied: haemoglobin concentration, haematocrit, number of red blood cells, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, blood pH, oxygen affinity of blood and stripped haemolysate, Root effect, the number of haemoglobins separated by polyacrilamide electrophoresis, iron concentration and osmotic pressure of the serum. Plagioscion squamosissimus , the only freshwater species studied, clearly separated from the other four species; it exhibited the highest haemoglobin number, haemotocrit, number erythrocytes, of oxygen affinity of the haemolysate, and the lowest oxygen affinity of the blood, iron concentration, pH and osmotic pressure.     

Radiation induced structural and functional modification of haemoglobin

An abnormality in the primary structure of dog haemoglobin was observed 1-20 days after their whole body irradiation with 190 keV X-rays (4.0 Gy). It consisted in a substitution of tryptophane residue in position 15 of the beta-chain for serine. The percentage of abnormal beta-chains in different time intervals after irradiation was determined. The structural changes have functional impact: an increasing haemoglobin affinity to oxygen. This could be explained on the basis of changes in the tertiary structure of haemoglobin, which may result from the substitution Trp 15----Ser15 in the beta-chain which may influence the haeme ability to bind oxygen.     

Effects of stopping smoking for 48 hours on oxygen availability from the blood: a study on pregnant women.

The effects of stopping smoking for 48 hours on factors governing the availability of oxygen from the blood--that is, carboxyhaemoglobin (COHb), haemoglobin-oxygen (HbO2) affinity, and haemoglobin concentration--were measured in women in the last trimester of pregnancy. Three groups were studied: smokers, smokers who stopped smoking for 48 hours, and non-smokers. The 22 smokers had higher initial COHb values and greater HbO2 affinity than the 10 non-smokers, but their total haemoglobin concentrations were also higher, so that their oxygen availability was not significantly reduced. In the 11 smokers who stopped the reduction in COHb and decrease in HbO2 affinity led to a significant increase of 8% in "available oxygen" in 48 hours. Since even small improvements in oxygen delivery to the tissues may confer critical benefit to the fetus, particularly during labour or when exposed to general anaesthesia, smoking should be discouraged for 48 hours before elective deliveries. The same consideration might reasonably be applied to patients undergoing general anaesthesia for all elective operations.     

Haemoglobin Hiroshima and the Mechanism of the Alkaline Bohr Effect

HAEMOGLOBIN Hiroshima is a variant with interesting physiological properties^1,2 discovered in a Japanese family. Its Bohr effect is halved, its oxygen affinity at physiological pH increased about three-fold and haem-haem interaction is somewhat reduced compared with normal haemoglobin. In 0.1 M NaCl solutions initially stripped of phosphate, 2,3-diphospho-glycerate (2,3-DPG) diminishes the oxygen affinity as in haemoglobin A (H. F. Bunn, unpublished results). The amino-acid substitution originally deduced for this abnormal haemoglobin was histidine 143 (H21)β ? aspartic acid¹. It was possible to conceive of a mechanism which accounted for its diminished Bohr effect³, but the normal response of its oxygen affinity to 2,3-DPG was inconsistent with the proposed role of histidine 143 in 2,3-DPG binding by haemoglobin A^4,5. An X-ray crystallographic study of deoxyhaemoglobin Hiroshima has now revealed that the replacement occurs not in position 143 but 146β. This was confirmed by chemical methods and the physiological properties of this haemoglobin are now satisfactorily accounted for. The results support the role of histidine 146β in the alkaline Bohr effect⁶.     

Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout.

Acclimation of rainbow trout to 5, 15 and 22 degrees C for periods exceeding 4 months had no significant effect on the oxygen affinity of whole blood or on the concentration of ATP, which is the main organic phosphate in red cells. Slight differences were, however, found in the oxygenation properties of the haemolysates, which correlate with changes in the relative concentration of the multiple haemoglobins. The oxygen-binding properties of the main haemoglobin components account for the observed differences in the haemolysates. The possible thermoacclimatory significance of changes in haemoglobin multiplicity and co-factor concentrations is discussed.     

Oxygen-binding and immunological properties of complexes between dextran and animal haemoglobins.

Complexes of dextran 20 000 with haemoglobins of sheep, rabbit, dog, bovine and human origin were prepared through alkylation of haemoglobin by N-bromoacetylaminoethylamino-dextran. The yields were uniformly high. Complex-formation in each case was accompanied by the disappearance of reactive thiol groups on the haemoglobin, and by an increase in the affinity of the haemoglobin for oxygen. The immunological properties of dog, rabbit and sheep dextran-haemoglobin were investigated in both homologous and heterologous species. The complexes were found to be non-immunogenic in the homologous species. In heterologous species the anti-haemoglobin response induced by each complex was generally of a similar level to that induced by the haemoglobin alone.     

Acid Bohr effect of a monomeric haemoglobin from Dicrocoelium dendriticum. Mechanism of the allosteric conformation transition

The dioxygen affinity of Dicrocoelium dendriticum haemoglobin was determined as a function of pH with a thin-layer diffusion technique. From the oxygen dissociation and association curves Hill coefficients h equal 1 were obtained throughout. Ultracentrifugation studies prove this haemoglobin to be monomeric irrespective of pH and ligation state. Thus, Dicrocoelium haemoglobin is a non-cooperative monomer. It has the highest O2 affinity so far known for any monomeric haemoglobin: its half-saturation pressure, p50 value, ranges at 25 degrees C from 0.016 mm Hg to 0.15 mm Hg (2.13-20.0 Pa) dependent on pH. Dicrocoelium haemoglobin shows an acid Bohr effect only and as such it constitutes a new class of haemoglobins. Its log p50 versus pH plot (Bohr effect curve) is characterized by a large amplitude, delta log p50 = 0.96, and an inflection point (Bohr effect pK) at pH 5.0. A model for the acid Bohr effect of D. dendriticum haemoglobin is proposed. By generalization, both the alkaline and the acid Bohr effect in various monomeric haemoglobins may arise from a single Bohr group complex (salt bridge).     

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.     

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.     

Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.     

Effect of polyanionic polymers on haemoglobin oxygen-binding properties: application to the synthesis of covalent conjugates with low oxygen affinity

Polyanionic water-soluble polymers containing sulphate, phosphate and polycarboxylate groups were synthesized. These compounds, when simply added to haemoglobin solutions, were shown to lower the affinity of the protein for oxygen. Their influence on oxygen affinity was regarded as the result of a specific interaction of the polymer anionic groups inside the 2,3-diphosphoglycerate-binding site of deoxyhaemoglobin. On the other hand, these polymers were linked to deoxyhaemoglobin to give covalent conjugates also exhibiting an oxygen affinity lower than that of free haemoglobin in the presence of 2,3-diphosphoglycerate, its natural effector, which means that after fixation, the polyanionic polymers are still acting as effectors.     

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.     

Simultaneous occurrence of Hb C trait and polycythaemia vera

A slow abnormal haemoglobin was found in a 27 year-old Negro man who had polycythaemia vera. Chemical and structural analysis showed it to be Hb C. The oxygen affinity showed a normal P50 value. Clinical and haematological investigations are described and discussed.