Blood oxygen equilibria and theoretical models. I. Effect of protons in trout (Salmo gairdneri) and human red cells, in absence of organic phosphates

1. Oxygen equilibrium curves were measured on red cells that had been depleted of organic phosphates, for rainbow trout red cells between pH 7-9, at 15 and 20 degrees C, and for human red cells between pH 6.8-8.0, at 37 degrees C. 2. The data were fitted to the models of Adair and of Monod et al. (MWC model). Parameters were estimated by the non-linear least-squares method, from which the number of Bohr protons released during oxygenation was calculated. 3. For trout as for human red cells, the pH affects the first step of oxygenation and the overall oxygenation in nearly equal proportion. For human red cells, the association constant for binding of the last oxygen, expressed by k4 or kR, is not affected by pH, indicating that the R structure is free of constraints. For trout red cells, there is a pronounced pH dependence of this constant at low pH, which is about 10-fold increased between pH 7-9. This corresponds to the Root effect that impairs the T----R transition, and confirms previously published data for normal trout and human red cells. 4. For trout red cells, small functional heterogeneity is evidenced, despite Hb multiplicity, and effect seems linked to the level of temperature. 5. The specific effect of organic phosphates, evident for human red cells, contrasts with a lack of effect for trout red cells, when taking into account the intracellular pH values.     

The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii)

• 1.1. Oxygen equilibrium curves were measured on trout red blood cell suspensions at pH 7.8 and 8.4 at 15, 20 and 25 C. Normal red cells and red cells that had been depleted of their ATP content were used.
• 2.2. The equilibrium data were fitted to the Adair's model and the enthalpy (ΔH) and entropy (ΔS) changes for the first and fourth steps of oxygenation and for overall oxygenation were calculated from the temperature dependencies of the Adair constants.
• 3.3. For normal red blood cells, the apparent heat for the first oxygenation step, δh₁, is close to zero.
• 4.4. Temperature insensitivity of this step at physiological pH, combined with a large pH dependence, probably denotes a property of Hb4, the Root effect Hb of trout blood.
• 5.5. At pH 7.8, ΔH₄ is about —4kcal/mol, a small value which may be attributed to the large release of Bohr protons that occurs at the last oxygenation step and corresponds to an endothermic process which opposes to the exothermic oxygenation of the haem.
• 6.6. The ΔH₄ value appears to have a large influence on the enthalpy for overall oxygenation.
• 7.7. Results for ATP-free red cells are consistent with a mere increase in the intracellular pH and suggest that ATP has no specific effect at and above pH_i ~ 7.7.
• 8.8. Effects of temperature and pH on trout red blood cell isotherms emphasize the primary importance of the major component of trout blood, namely Hb4, in trout blood functional properties.

     

An electrolytic method for determining oxygen dissociation curves using small blood samples: the effect of temperature on trout and human blood.

1. A detailed account is given of an electrolytic method for determining the oxygen dissociation curve of fish blood using a single sample of 50-100 mul for the whole curve. The accuracy and some of the problems arising from its uses are discussed. 2. Oxygen dissociation curves have been determined for trout blood and human blood at temperatures of 15 and 37 degrees C. The relationship between P50 and temperature is similar to that obtained using other methods. Absolute values of P50 are generally lower than those obtained by other methods, especially in the case of fish blood. 3. The effect of PCO2 and pH on the oxygen dissociation curve of trout blood is tested and it is shown that PCO2 has a more marked effect than pH when the other factor is maintained at a constant level. The Bohr factor (delta log P50/delta pH) appears to be approximately the same and independent of the PCO2. 4.The P50 of ray blood determined from fish during and after an operation showed an increased Bohr factor.     

The oxygen uptake of Sarotherodon niloticus L. and the oxygen binding properties of its blood and hemolysate (Pisces: Cichlidae)

The oxygen consumption of Sarotherodon niloticus L. was found to decline below a critical oxygen concentration of about 2 mg O2/l. An important influence of CO2 on the oxygen affinity of whole blood was observed at all temperatures between 20 and 35 degrees C for gas mixtures containing 5.6% CO2. Purified hemolysate showed extremely high oxygen affinities (p50 = 1.08 mmHg at pH 8.2 and 20 degrees C). Low cooperativity was observed at all temperatures from 20 to 35 degrees C, and pH values between 6.5 and 8.2. The Bohr effect proved to be important at pH values lower than pH 7.5 (phi = delta log P50/delta pH = -0.58 between pH 6.5 and 7.0 at 35 degrees C). The oxygen affinities show high thermal sensitivity without a marked pH influence (delta H value for overall oxygenation at pH was -71.7 kJ/mol). The obtained results are interpreted as adaptations to diurnal variations in ambient temperature and oxygen availability.     

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.     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Human whole-blood O2 affinity: effect of CO2

The proton Bohr factor (phi H = alpha log PO2/alpha pH), the carbamate Bohr factor (phi C = alpha log PO2/alpha log PCO2), the total Bohr factor (phi HC = d log PO2/dpH[base excess) and the CO2 buffer factor (d log PCO2/dpH) were determined in the blood of 12 healthy donors over the whole O2 saturation (SO2) range. All three Bohr factors proved to be dependent on SO2, although to a lesser extent than reported in some of the recent literature. At SO2 = 50% and 37 degrees C, we found phi H = -0.428 +/- 0.010 (SE), phi C = 0.054 +/- 0.006, and phi HC = -0.488 +/- 0.007. The values obtained for phi H, phi C, and d log PCO2/dpH were used to calculate phi HC. Calculated and measured values of phi HC proved to be in good agreement. In an additional series of 12 specimens of human blood we determined the influence of PCO2 on phi H and the influence of pH on phi C. At SO2 = 50%, phi H varied from -0.49 +/- 0.009 at PCO2 = 15 Torr to -0.31 +/- 0.010 at PCO2 = 105 Torr and phi C from 0.157 +/- 0.015 at pH = 7.80 to 0.006 +/- 0.009 at pH = 7.00. When on the basis of these data a second-order term is taken into account, a still slightly better agreement between measured and calculated values of phi HC can be attained.     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

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.     

Non-cooperative oxygen binding in the erythrocytes of pre-implanted sheep embryos

The whole-blood oxygen equilibrium curve in sheep embryos at 17 days gestation was essentially hyperbolic, indicating non-cooperative O2 binding with Hill's coefficient, n approximately equal to 1.2. O2 affinity was higher in embryonic blood (P50 = 7.1 mmHg at pH = 7.4 and 39 degrees C) than in maternal blood (P50 = 32.6 mmHg at pH = 7.4 and 39 degrees C). The Bohr effect was apparently smaller in the embryo (delta log P50/delta pH = --0.52) than in maternal blood (--0.36).     

Temperature- and pH-dependence of the oxygen-binding reaction of human fetal haemoglobin.

O2 binding to human haemoglobin F0 was studied at high haem concentrations (3 mM) in the temperature range 15-35 degrees C and in the pH range 6.8-8.7 at 25 degrees C. Comparison with O2 binding to human adult haemoglobin A0 under identical solution conditions reveals striking similarities in the Bohr effect and the enthalpy of oxygenation between the two haemoglobins.     

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.     

The evolution of Root effect hemoglobins in the absence of intracellular pH protection of the red blood cell: insights from primitive fishes

The Root effect, a reduction in blood oxygen (O₂) carrying capacity at low pH, is used by many fish species to maximize O₂ delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH_i) protection mechanism of more derived species’ red blood cells (i.e., adrenergically activated Na⁺/H⁺ exchangers; βNHE). These basal actinopterygians may consequently experience a reduction in blood O₂ carrying capacity, and thus O₂ uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O₂, i.e., Hb O₂ desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH_i 7.15–7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell βNHE activity, it is unlikely that the possession of a Root effect would impair O₂ uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived teleosts studied to date. This is paralleled by the initial appearance of the choroid rete in bowfin, as well as a significant decrease in Hb buffer value and an increase in Bohr/Haldane effects, together suggesting bowfin as the most basal species capable of utilizing its Root effect to maximize O₂ delivery to the eye.     

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).     

Effect of Cl- and H+ on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes

Bovine hemoglobin was cross-linked with glutaraldehyde, resulting in high oxygen affinity polymeric hemoglobin dispersions of varying molecular weight distributions. High oxygen affinity acellular oxygen carriers were designed in order to exhibit oxygen release profiles closer to that of human red blood cells (RBCs), without exhibiting the inherent increased vasoactivity that occurs with low oxygen affinity acellular oxygen carriers (1, 2). Oxygen dissociation curves were measured for polymerized hemoglobin dispersions at various pH values (7.0, 7.4, and 8.0) and chloride ion concentrations. Unmodified hemoglobin showed an increase in oxygen affinity with increased chloride ion concentration and a decrease in oxygen affinity with increased pH, as was previously demonstrated in the literature (3). For glutaraldehyde-polymerized hemoglobin dispersions, the ability of the oxygen affinity to respond to changes in Bohr H+ and Cl- concentration was weakened. However, at acidic physiological pH (pH = 7), the Bohr effect was still present at high Cl- concentrations. Thus, the Bohr effect maintained some dependency on the Cl- concentration.     

Blood oxygen equilibria and theoretical models. II. A critical estimation

1. Theoretical models can fit the oxygen equilibria of trout and human red cell suspensions, and describe the apparent oxygenation process in the red cell. 2. The assumption that full oxygenation is attained at atmospheric O2 pressures can result in biphasic Hill plots and high Hill coefficients for high O2 saturations. This phenomenon must not be confused with aggregation of hemoglobin. 3. Problems specific to measurements of red cell suspensions, regarding the ionic cellular composition and its stability with time, are approached. Changes of buffer osmolarity, and--for trout--addition of adrenaline, within physiological proportions, have no impact on the results. 4. This tends to validate the general significance of equilibrium data obtained on this material, regarding the effects of protons and organic phosphates, although complex ionic movements across the red cell membrane are known to occur in the animal under certain circumstances.     

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.     

PH dependence of the Adair constants of human hemoglobin. Nonuniform contribution of successive oxygen bindings to the alkaline Bohr effect.

In order to solve the problem of an apparent discrepancy between the pH variance of oxygen equilibrium curve and the linear relation between the number of released Bohr protons and the degree of ligation, precise oxygen equilibrium curves of human hemoglobin were determined at a number of pH values from 6.5 to 8.8. From the equilibrium data individual steps (Adair constants), ki (i equals 1, 2, 3, 4), were obtained and the number of Bohr protons (deltaHi+) released on the ith stage of oxygenation was estimated. The pH dependence of k4 was very small, while the other ks strongly depended on pH over the pH range examined. As a consequence, the contribution of each step of oxygen binding to the alkaline Bohr effect nonuniform: deltaH4 was very small compared with deltaH1+, deltaH2+, and deltaH3+. In spite of this, calcuation has shown that the fractional number of released protons is essentially proportional to fractional oxygen saturation because of cooperative effects in hemoglobin. Thus, the present study indicates that the linear relationship between the fractional number of released protons and the degree of ligation, as obtained from titration experiments, is not necessarily incompatible with the pH variance of the shape of the oxygen equilibrium curve. The nonuniform pH depencence of the Adair constants implies that the two-state allosteric model of Monod, J., Wyman, J., and Changeus, J.P. (1965) J. Mol. Biol. 12, 88-118 is not adequate to describe the heterotropic effect caused by protons.