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of phosphate, 2,3-diphospho-glycerate (2,3-DPG) diminishes the oxygen affinity as in haemoglobin A (H. F. Bunn, unpublished results).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.     

The interaction between oxygen and carbon dioxide movements in fishes

Many teleost fishes have haemoglobins which possess a Root effect, a large Haldane effect and a low buffer capacity. This combination of characteristics influences the interaction between movements of oxygen and carbon dioxide in the red cell, in the respiratory epithelium, and in the tissues. The presence of the Root effect may limit oxygen uptake at the gills due to an accumulation of Bohr protons released upon oxygenation. However, the Root effect is probably important in maintaining or elevating blood PO₂ during muscle capillary transit, enhancing oxygen delivery to the tissues.Bohr protons are reversibly bound to haemoglobin. The release of Bohr protons during oxygenation facilitates bicarbonate dehydration at the gills, while Bohr proton binding facilitates CO₂ hydration at the tissues. In some teleost fishes, most of the Bohr protons are released and bound to haemoglobin, between 50 and 100% of haemoglobin-oxygen saturation (27). This trait is probably significant in maximizing oxygen uptake at the gills and in conserving body CO₂ stores during exposure to hypoxia and exercise, when the lower reaches of the haemoglobin-oxygen equilibrium curve are used.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.     

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

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

Studies on the reaction of isocyanides with haemproteins. I. Equilibria and kinetics of the binding to the isolated chains of human haemoglobin

The equilibrium and kinetics of a series of alkylisocyanides (methyl, ethyl, isopropyl and tertiary butyl) reacting with the isolated chains of human haemoglobin (both in their SH and p-mercuribenzoate forms) has been investigated; the results lead to the following conclusions.

1.

(a) The reactions resemble, in their general properties, those with other ligands, such as O₂ or CO; there is absence of homotropic interactions (n ~ 1), and of heterotropic interactions (no Bohr effect) and a much higher equilibrium constant (I) than for intact haemoglobin, reflected kinetically in a large increase in the “on” constant (í).     

Functionally distinct haemoglobins of the cryopelagic Antarctic teleost Pagothenia borchgrevinki

Pagothenia borchgrevinki , has a higher haemoglobin concentration than other Antarctic notothenioids and the high oxygen capacity may correlate with the relatively active mode of life of this fish. The fish has five haemoglobins (Hb C, Hb 0, Hb 1, Hb 2 and Hb 3) with Hb 1 accounting for 70–80% of the total, and Hb C being present in trace amounts. Hb 1 and Hb 2 are functionally similar in terms of Bohr and Root effects. Hb 3 has a weaker Bohr effect than Hb 1 and Hb 2, and the Root effect is similar to that of Hb 1. Hb 0 has a strong Bohr effect and the Root effect is enhanced to a larger extent by the physiological effectors chlorides and phosphates than that of the other components with the exception of Hb C. The heats of oxygenation are lower than those of temperate fish haemoglobins. Temperature variations may have a different effect on the functional properties of each haemoglobin, and chloride and phosphates may play an important role in the conformational change between the oxy and deoxy structures. The complete amino acid sequences of Hb 1 and Hb 0, as well as partial N-terminal or internal sequences of the other haemoglobins, have been established. The high multiplicity of functionally distinct haemoglobins indicates that P. borchgrevinki , has a specialized haemoglobin system.     

Sequence analysis and tissue expression of a non-Bohr beta-globin cDNA from Atlantic salmon

The presence of a haemoglobin protein which does not exhibit a Bohr effect has been found only in fish living in fast flowing waters. We report the cloning of the first non-Bohr effect beta-globin cDNA from an adult Atlantic salmon kidney bank. Nucleotide sequence analysis of this cDNA shows that the predicted beta-globin peptide comprises 147 amino acids with a calculated molecular mass of 15 975 Da and an overall amino acid homology of 40 to 50% to higher vertebrates and 60–90% to fish sequences. This sequence confirms the important amino acid residues which are changed thus causing loss of the Bohr effect [Powers, D.A. and Edmunson, A.B. (1972) Multiple hemoglobins of catostomid fish. J. Biol. Chem. 247, 6686–6693; Brunori, M. (1975) Molecular adaptation to physiological requirements: the hemoglobin system of trout. Curr. Topics Cell. Regul. 9, 1–39]. This loss allows the haemoglobin protein to have a higher oxygen affinity, as it does not release oxygen when the pH of the surrounding environment decreases, which is an important ability for the fish in times of stress.     

Osmoregulatory bicarbonate secretion exploits H+-sensitive haemoglobins to autoregulate intestinal O2 delivery in euryhaline teleosts

Marine teleost fish secrete bicarbonate (HCO₃ ^?) into the intestine to aid osmoregulation and limit Ca²⁺ uptake by carbonate precipitation. Intestinal HCO₃ ^? secretion is associated with an equimolar transport of protons (H⁺) into the blood, both being proportional to environmental salinity. We hypothesized that the H⁺-sensitive haemoglobin (Hb) system of seawater teleosts could be exploited via the Bohr and/or Root effects (reduced Hb-O₂ affinity and/or capacity with decreasing pH) to improve O₂ delivery to intestinal cells during high metabolic demand associated with osmoregulation. To test this, we characterized H⁺ equilibria and gas exchange properties of European flounder (Platichthys flesus) haemoglobin and constructed a model incorporating these values, intestinal blood flow rates and arterial–venous acidification at three different environmental salinities (33, 60 and 90). The model suggested red blood cell pH (pH_i) during passage through intestinal capillaries could be reduced by 0.14–0.33 units (depending on external salinity) which is sufficient to activate the Bohr effect (Bohr coefficient of ?0.63), and perhaps even the Root effect, and enhance tissue O₂ delivery by up to 42 % without changing blood flow. In vivo measurements of intestinal venous blood pH were not possible in flounder but were in seawater-acclimated rainbow trout which confirmed a blood acidification of no less than 0.2 units (equivalent to ?0.12 for pH_i). When using trout-specific values for the model variables, predicted values were consistent with measured in vivo values, further supporting the model. Thus this system is an elegant example of autoregulation: as the need for costly osmoregulatory processes (including HCO₃ ^? secretion) increases at higher environmental salinity, so does the enhancement of O₂ delivery to the intestine via a localized acidosis and the Bohr (and possibly Root) effect.     

Oxygenation properties of haemoglobins from the flatfish plaice (Pleuronectes platessa) and flounder (Platichthys flesus)

Summary The oxygen binding properties and some haematological data of haemoglobins of plaice (Pleuronectes platessa) and flounder (Platichthys flesus) were measured and compared, with the view of discerning their adaptations to the benthic habitat.Compared to plaice, flounders posses higher haematocrit and haemoglobin levels, and their haemoglobin has a higher oxygen affinity, smaller Bohr and Root effects and appears to have a greater ATP sensitivity. At pH 7.6, the half-saturation oxygen tensions,P ₅₀, of stripped plaice and flounder haemoglobins amount to about 5.9 and 4.2 torr, and the Bohr shifts (logP ₅₀/ pH) to –0.51 and –0.34, respectively. The species differences in the oxygen binding patterns occur after stripping the haemoglobin in solution, from dissolved ions and thus appear to be inherent in the pigment molecules. The differences suggest that the haemoglobin of flounder is better adapted to hypoxic and hypercarbic environments than plaice haemoglobin, in accordance with the more inshore occurrence of the former species.The molar ratios of ATP to haemoglobin tetramers are low in plaice and flounder (about 1.6); in plaice this ratio corresponds to that where the cofactor effect is most pronounced. In contrast to previous evidence for an adaptive reduction in the temperature dependence of the haemoglobin of flounder but not of plaice from the Baltic Sea (Friedrich, 1935), the same temperature effects were found in representatives of the two species both from the Dutch North Sea and the Danish Kattegat.     

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).     

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.     

Oxygen-binding characteristics of three extra-cellular haemoglobins of Artemia salina

The oxygen-binding characteristics of the three extracellular haemoglobins of brine shrimp (Artemia salina) were studied in vitro by using highly purified preparations. Haemoglobin I is induced last in the development of brine shrimps when functional gills are formed. It has the lowest oxygen affinity (p₅₀ 5.34mmHg), an intermediate Bohr effect (ø −0.09 at 20°C) above pH8 and a temperature-sensitivity (ΔH −44.8 to −45.6kJ/mol at pH8–9) comparable with those observed with other invertebrate haemoglobins [Weber & Heidemann (1977) Comp. Biochem. Physiol. A 57, 151–155]. Haemoglobin II, which is the first to be induced, soon after hatching of nauplius larvae, persists generally throughout the whole adult life. It has an intermediate oxygen affinity (p₅₀ 3.7mmHg), the highest Bohr effect (ø −0.21 at 20°C) above pH8 and a similar temperature-sensitivity (ΔH −46.0 to −54.8kJ/mol at pH8–9) as haemoglobin I. However, haemoglobin III, which is induced second several hours after the induction of haemoglobin II but disappearing from the haemolymph in the middle of adult life, has the highest oxygen affinity (p₅₀ 1.8mmHg), the lowest Bohr effect (ø −0.03 at 20°C) above pH8.5 and a high resistance against temperature variation between 10 and 25°C at pH8.5–9 (ΔH −22.6 to −23.0kJ/mol). At pH7.5–8, haemoglobin III exhibits a similar temperature-sensitivity under 30°C as do other haemoglobins. All three haemoglobins have a rather low co-operativity, with Hill coefficients (h 1.6–1.9 at pH8.5), which are dependent on both pH and temperature. The highest co-operativity was observed at 20°C and pH9 for haemoglobins I and II, whereas it was at 27°C and pH8.5 for haemoglobin III. Thus the oxygen-binding behaviour of haemoglobin III in vitro is significantly different from those of haemoglobins I and II and indicates possibly its specific physiological role in vivo in the adaptive process in the natural environment.     

The haemoglobin system of the mudfish, Labeo capensis: adaptations to temperature and hypoxia

The effect of temperature and hypoxic acclimation on the haemoglobin system and intraerythrocytic organic phosphate concentrations in the South African mudfish, Labeo capensis, have been investigated. Exposure to hypoxia or increased temperature raised haemoglobin concentration and decreased NTP/Hb ratio. Temperature acclimation did not effect the oxygenation characteristics of the haemolysate or haemoglobin multiplicity, as evident from isoelectric focussing experiments that showed one cathodic (Hb I) and three anodic haemoglobins (Hb II, III and IV). Oxygen equilibria of the isolated components showed a smaller Bohr effect and lower temperature and organic phosphate sensitivities in the cathodic than in the anodic haemoglobins. Unlike the trout and eel haemoglobin systems, both the anodic and cathodic haemoglobins from L. capensis exhibited sensitivity to organic phosphates but the effect was smaller in the latter. The results indicate that oxygen transport in mudfish blood is supported by variations in the red cell organic phosphate\haemoglobin ratio and the functional differentiation between anodic and cathodic haemoglobins.     

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

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

Mechanism of charge compensation and impairment of co-operative functions in haemoglobin Tacoma (Arg B12(30)beta leads to Ser).

A difference Fourier synthesis of deoxyhaemoglobin Tacoma minus deoxyhaemoglobin A at 3.5 Å resolution has been calculated. The map shows a large negative peak due to the removal of the guanidinium group of Arg B12(30)β, surrounded by positive and negative peaks indicative of some atoms moving towards, and others away from, the vacated site. Among the latter, the most important is the carboxylate of Glu B8(26)β which is hydrogen-bonded to the guanidinium group of the arginine in haemoglobin A, but swings round its α-β carbon bond towards the imidazoles of histidines G18(116) and 19(117)β in haemoglobin Tacoma. This movement would raise the pK values of the histidines, so that their positive charges compensated for the loss of the arginine. This may explain why haemoglobin Tacoma has the same electrophoretic mobility as haemoglobin A. It is shown that haemoglobin Tacoma has a lower oxygen equilibrium constant K_T and a larger allosteric constant L than haemoglobin A. The lowering of K_T may be due to the loosening of the T structure in haemoglobin Tacoma consequent upon the removal of the hydrogen bonds made by the guanidinium group of Arg B12(30)β at the α₁β₁ contact. Their removal also accounts for the decreased stability of haemoglobin Tacoma. We cannot yet explain its diminished Bohr effect, nor the increase in L.     

Respiratory characteristics of the blood pigments of three worms from an intertidal mudflat

Abstract

Three worms living in an intertidal mudflat near Auckland have respiratory pigments with different oxygen-binding properties. The burrowing sipunculid Xenosiphon mundanus has hemerythrin contained within coelomic cells. Oxygen is co-operatively bound to the hemerythrin (Hill's coefficient, n, = 1.8), resulting in a sigmoidal oxygen-binding curve of high oxygen affinity (halfsaturation tension, P₅₀, = 7.0 mm Hg at pH 7.5 and 20°C). Oxygen release is regulated by pH, and the Bohr effect is quantified by Ф = Δ log P₅₀ / ΔpH = - 0.30. The errant polychaete Glycera sp. has hemoglobin-charged coelomocytes with low oxygen affinity (P₅₀ = 13.5 mm Hg at pH 7.3 and 20°C)? and the oxygen-binding curve is essentially hyperbolic and insensitive to pH. The burrowing polychaete Abarenicola affinis has a vascular (circulating) erythrocruorin which binds and releases much oxygen for small changes in PO₂ (Hill's n = 3.8) and is insensitive to pH (Ф = -0.09). The physiological properties of these pigments cannot be related to the availability of oxygen in the near-environment, or to the habits of the animals, but appear to be dictated by the level of body organisation, particularly with regard to the gas exchange surfaces.     

Respiratory properties of the blood of the burrowing red band fish Cepola rubescens L.

The respiratory properties of the whole blood of the burrowing red band fish Cepola rubescens L. were investigated. Oxygen dissociation curves constructed at 15°C were found to be close to hyperbolic in shape with a mean value for the cooperativity coefficient at half-saturation (n₅₀) of 1.56. Half-saturation oxygen tension (P₅₀) for pH = 7.56 (mean in vivo pH of venous blood) was 27 Torr. The blood showed a marked Bohr effect ($\text{Δ log}\text{P}{}_{50}\text{ΔpH}\text{= ?1.19}$) and also a Root effect which at the in vivo pH reduced oxygen carrying capacity by 20%. The Pv_CO2 was 3.2 Torr and the buffering power of the blood was low, the buffer value of true plasma averaging 5.43 mmol · 1^?1 · pH^?1. It is suggested that the large Bohr effect coupled with the low buffer value confers on the haemoglobin a flexibility, in terms of oxygen affinity, to withstand changes which occur in environmental oxygen tensions.     

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.     

The blood oxygen binding properties of hypoxicSalmo gairdneri

Summary The blood oxygen binding properties of rainbow trout responded to environmental hypoxia (the oxygen saturation of water 30% at 11°C) in three ways. The quickest response was a moderate acidosis, leading to slightly lowered blood oxygen loading due to the Bohr effect. The second response, an increase of blood oxygen carrying capacity, was completed with 6 h from the onset of hypoxia. The speed of the response suggests that the formation of new haemoglobin played no practical role, the increase being caused either by a decrease of plasma volume or the liberation of erythrocytes from a storage organ. The slowest response, a 25% increase of the blood oxygen affinity within a week of hypoxia, was probably caused by the concurrent decrease of the erythrocyte ATP concentration from 4.45 to 2.51 mol/ml erythrocytes.     

Biochemical and functional characterization of Rapana thomasiana hemocyanin

• 1.1. The hemocyanin (Hc) of the marine gastropod mollusc Rapana thomasiana was collected from animals living on the west coast of the Black Sea and characterized for its biochemical and functional properties.
• 2.2. This Hc is very similar to other gastropod Hcs as far as amino acid composition, general structure and reactivity of the binuclear copper active site are concerned.
• 3.3. Some peculiarities in the dissociation-reassociation pattern are observed in comparison to other gastropod Hcs, in particular with respect to the ability to form sopramolecular aggregates.
• 4.4. Changes in pH disclose a strong reverse Bohr effect. Different R and T states are required to describe the oxygen binding curves at the different pHs.