A fetal-maternal shift in the oxygen equilibrium of hemoglobin from the viviparous caecilian,Typhlonectes compressicauda

• 1. The equilibria and kinetics of oxygen binding by blood and hemoglobin from adult and fetal caecilians,Typhlonectes compressicauda, have been measured.
• 2. The oxygen affinity of fetal blood is higher than that of adult blood.
• 3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each.
• 4. Adult and fetal hemoglobins have identical oxygen equilibria. Stripped hemoglobins have a high oxygen affinity and no Bohr effect between pH 6.5 and 10.0. An “acid”, reversed Bohr effect is present below pH 6.5. The addition of 1 mM ATP reduces the oxygen affinity markedly and produces a moderate, normal Bohr effect.
• 5. The major nucleoside triphosphate in fetal and adult erythrocytes is adenosine triphosphate: about 10% of the nucleoside triphosphates is guanosine triphosphate. Adult erythrocytes contain 3 times as much ATP as do the fetal erythrocytes.
• 6. The fetal to maternal shift in the oxygen equilibrium is mediated entirely by the difference in ATP content of the maternal and fetal red blood cells.

     

Equilibria and kinetics of oxygen and carbon monoxide binding to the haemoglobin of the teleostSynbranchus marmoratus

• 1. The haemoglobins of the air-breathing fishSynbranchus marmoratus have multiple components and demonstrate polymorphism.
• 2. The equilibrium of oxygen with whole cell and isolated haemoglobin has been compared. There is a considerable alkaline Bohr effect and this is more marked in stripped haemoglobin to which 1 mM ATP has been added.
• 3. The kinetics of oxygen dissociation from oxyhaemoglobin and of carbon monoxide combination to deoxyhaemoglobin have been studied, and show surprisingly uniform kinetic time courses for a system composed of multiple components.
• 4. These findings are discussed within the framework of the requirements of the animal in its mixed aerial-aquatic environment.

     

Functional studies on the separated hemoglobin components of an air-breathing catfish,Hoplosternum littorale (Hancock)

• 1. The two hemoglobins, Hb I and II, of the obligate air-breathing catfish,Hoplosternum littorale have been isolated.
• 2. The unfractionated stripped hemoglobin has a high oxygen affinity, a normal alkaline Bohr effect, and a Root effect.
• 3. Both the Bohr and Root effects are enhanced by 1 mM ATP.
• 4. Stripped Hb I has a relatively high oxygen affinity, a reversed Bohr effect between pH 6.0 and 8.0 (Δlog P₅₀2DpH> 0), but no Root effect. Addition of 1 mM ATP to Hb I causes a marked reduction in the oxygen affinity, a change to a normal alkaline Bohr effect (Δlog P₅₀ΔpH< 0), but no Root effect.
• 5. Stripped Hb II has a lower oxygen affinity at low pH and a higher oxygen affinity at high pH than does Hb I. Hb II shows a large alkaline Bohr effect which is only slightly increased by 1 mM ATP and a Root effect at low pH which is enhanced by 1 mM ATP.
• 6. The observed rates of O₂ dissociation and of CO combination with Hbs I and II show differences which parallel those observed in the oxygen equilibrium measurements.

     

Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

• 1.1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components.
• 2.2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation.
• 3.3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect.
• 4.4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity.
• 5.5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

     

The isolation and characterization of the hemoglobin ofBrachyplatystoma sp.: A tropical catfish

• 1. The single hemoglobin component ofBrachyplatystoma sp. has been isolated. The CO-hemoglobin has an apparent molecular weight of 69,000 as determined by gel filtration.
• 2. The hemoglobin displays both acid and alkaline Bohr effects, as organic phosphate effect and no Root effect. The whole bloodp_1/2 for oxygen shifts from 10.7 mm Hg in air equilibrated solutions to 25.1 mm Hg after the addition of 5.6% CO₂ to the equilibration gas. Thep_1/2 of purified hemoglobin varies from 0.3 mm Hg at pH 8.4 to 4.5 mm Hg at pH 5.9. The Bohr effect measured for stripped hemoglobin between pH 8.0 and 7.0 isΔlog p_1/2/ΔpH= −0.23. Additions of 1 mM ATP induce a shift in the Bohr effect toΔlog p_1/2/ΔpH= −0.58 over the same pH range.
• 3. Then value of stripped hemoglobin solutions varies from 1 at pH 5.9 to 1.7 at pH 7.0. Additions of 1 mM ATP shift the variation inn to higher pH values, and cause an increase in then value (n = 2 at pH 7.4).
• 4. The kinetics of carbon monoxide binding and oxygen dissociation are pH dependent. The CO_on rate becomes autocatalytic as the pH is lowered, indicating positive subunit interactions. The O_2off rate was homogeneous at all pH values.
• 5. The Bohr effects ofBrachyplatystoma hemoglobin and other pimelodid hemoglobins are greater than those determined for the unfractionated hemoglobins of more sedentary species from other catfish families such as the Loricariidae and Callichthyidae.

     

Effect of pH on the kinetics of oxygen and carbon monoxide reactions with hemoglobin from the air-breathing fish,Loricariichthys

• 1.Loricariichthys sp., an air-breathing fish from the Amazon River has one major hemoglobin component.
• 2. Quantitative studies on the kinetics of O₂ dissociation and CO combination to the protein were performed by stopped-flow experiments at different pH values and a constant ATP concentration of 1.25 mM.
• 3. The oxygen dissociation shows a simple first order behavior and a strong pH dependence.
• 4. The CO combination kinetics, on the other hand, were homogeneous and fast at higher pH values and slow and clearly autocatalytic at pH values below 7.0.

     

Oxygen binding of intact coelomic cells and extracted hemoglobin of the echiuran Urechis caupo

• 1.1. The oxygen affinity of Urechis caupo coelomic cells is the same in normoxic and in hypoxic animals. There is no Bohr effect between pH 6.8 and 8.0.
• 2.2. The oxygen affinity of intact coelomic cells is the same as that of extracted, stripped hemoglobin. The oxygen binding properties of stripped hemoglobin are not affected by 1 mM ATP, IMP, or hydrogen ions between pH 6.8 and 8.0, nor do they clearly show cooperativity. The heat of oxygenation. ΔH, = −13.1 kcal/mol between 10 and 25 C.
• 3.3. Although U. caupo coelomic cell hemoglobin is tetrameric and intracellular, it apparently exhibits neither heterotropic nor homotropic interactions.

     

Kinetics of oxygen and carbon monoxide binding to the hemoglobins of the water snakes Liophis miliaris and Helicops modestus

• 1.1. Liophis miliaris and Helicops modestus are water snakes having different respiratory adaptiations to their specific habitats. L. miliaris is more active and spends more time on land than H. modestus. Knowledge of the equilibrium and kinetics of ligand binding to their hemoglobins leads to better understanding of molecular aspects of this adaptation.
• 2.2. Both snakes contain several hemoglobin types in their blood. Studies on the kinetics of oxygen dissociation and carbon monoxide combination with these hemoglobins were performed by stopped-flow and flash-photolysis experiments at various pH values, both in the presence and absence of adenosine triphosphate.
• 3.3. The oxygen dissociation kinetics of L. miliaris hemoglobins show a strong pH dependence and cooperative interactions between chains are indicated by autocatalytic time-courses at pH 7.0. In contrast, H. moledstus hemoglobins show nearly pH independent rate constants for oxygen dissociation and cooperative interactions between chains were not apparent. The hemoglobins of H. modestus show increased pH dependence in the presence of adenosine triphosphate.
• 4.4. The carbon monoxide combination kinetics differ for the hemoglobins of L. miliaris and H. modestus in general agreement with the differences found in the kinetics and equilibria of oxygen binding. Both the kinetic and steady-state difference between these hemoglobins may be advantageous in light of the behavioral differences of these two water snakes.

     

The measurement of the Bohr effect of fish hemoglobins by gel electrofocusing

• 1. Hemolysates from 16 species of Amazon fish and one amphibian were analyzed by gel electrofocusing. The change in isoelectric point upon deoxygenation provided a reliable estimate of the Bohr effect.
• 2. Certain species of fish had single hemoglobin components whose pI increased significantly upon deoxygenation, as in man. Other fish had hemoglobins whose isoelectric points were unaffected by deoxygenation. Six species of fish had at least two hemoglobin components, one of which had a reduced isoelectric point upon deoxygenation indicating a reversed Bohr effect, whereas the other(s) had an increased isoelectric point on deoxygenation, as occurs with the normal alkaline Bohr effect.
• 3. A close correlation was found between the change in isoelectric point with deoxygenation and the Bohr effect determined by oxygen equilibrium measurements.

     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.

     

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.

     

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.

     

Properties of hemocyanins isolated from Amazon river arthropods and molluscs

• 1. Hemocyanins from four organisms inhabiting the Amazon River were isolated and partially characterized.
• 2. Three arthropodan species (Dilocarcinus pagei cristatus, Silviocarcinus pardalinus andMacrobrachium amazonicum) possess hemocyanins whose subunit structure is remarkably simple. Regular and SDS polyacrylamide disc electrophoresis revealed predominantly single bands and no polymorphisms.
• 3. Oxygen-binding experiments showed that the three arthropodan hemocyanins possess large positive Bohr effects and pH dependence in the degree of subunit interaction.
• 4. The hemocyanin of one mollusc,Pila sp., was studied and its subunit size appears to be similar to that of other molluscan hemocyanins, i.e. a polypeptide of mol. wt 400,000. In the hemolymph,Pila hemocyanin probably exists as a mixture of 100 and 124S aggregates.
• 5. The oxygen binding properties of the large molecules ofPila hemocyanin are notable because of their low cooperativity and lack of a strong pH-dependence.

     

Oxygenation characteristics of the polymorphic hemoglobins of coho salmon (Oncorhynchus kisutch) at different developmental stages

• 1.1. The oxygen equilibria of the multiple hemoglobin of the fry and adult life stages of coho salmon (Oncorhynchus kisutch) were investigated. Components A 6–8 comprise 50–55% and >95% of the total hemoglobins of coho adults and fry respectively.
• 2.2. Fry hemoglobins exhibited a high oxygen affinity (P₅₀ = 3.9 mm Hg at 9.8°C, pH 8.5), a very large Bohr shift (ø = Δlog ₅₀/ΔpH = −1.729 at pH 7.1–7.5) which was non-linear in the pH range 6.8–8.5 and a large heat of oxygenation (ΔH = −20.8 kcal/mol).
• 3.3. Adult hemoglobins, however, exhibited a moderate oxygen affinity (P₅₀ = 14.0 mm Hg at 9.8°C, pH 8.2), a very small linear Bohr shift (ø = −0.172 at pH 7.0 −8.2) and a low heat of oxygenation (ΔH = −5.6- −7.5kcal/mol). Adenosine triphosphate had only minor influence on the oxygenation characteristics of adult heolyzates.
• 4.4. The results are discussed in terms of functional adaptation to environmental stresses and metabolic requirements.

     

Functional studies on the single component hemoglobin from an Amazon knife fish,Sternopygus macrurus

• 1. The whole blood of the non-air-breathing gymnotid teleost,Sternopygus macrurus, is half-saturated with oxygen at 5.2 mm Hg (apparent value) at 30°C in the absence of CO₂. Addition of 5.6% CO₂ causes the apparentP₅₀ value to increase over 3-fold.
• 2. The oxygen affinity of the stripped single-component hemoglobin at 20°C increases about 20-fold between pH 5.8 and 8.6 in the absence of ATP. This difference increases to 100-fold in the presence of 1 mM ATP.
• 3. A substantial Root effect is present: the stripped hemoglobin is only 70% saturated with O₂ at pH less than 6 when equilibrated with air.
• 4. The value of the Hill coefficient,n, is maximal near pH 7.0–7.5, and approaches 1.0 at high pH. The value is about 1.5 at low pH in the absence of ATP and 1.0 in the presence of 1 mM ATP.
• 5. The O₂ dissociation kinetics are heterogeneous at all pH values but most heterogeneous at low pH. The rate increases substantially as the pH decreases.
• 6. The CO combination kinetics as measured by the stopped flow technique are largely homogeneous except at high pH, but the CO combination kinetics after flash photolysis are markedly heterogeneous.

     

Studies of the functional properties of the hemoglobins ofHoplias malabaricus andHoplerythrinus unitaeniatus

• 1. Hemolysates fromHoplias malabaricus andHoplerythrinus unitaeniatus show blurred hemoglobin patterns with three and four bands, respectively, by alkaline disc gel electrophoresis.
• 2. The oxygen affinity of the stripped hemoglobin fromHoplerythrinus is about a third of that fromHoplias; theP₅₀ value ofHoplias Hb is about 1.3 mm Hg (pH 6.9 and 20°C). The addition of 1 mM ATP lowers the oxygen affinity of each hemoglobin 2.6-fold.
• 3. Both hemoglobins show Root and Bohr effects;Δlog P₅₀ΔpH= −0.40 for a stripped hemoglobins for the interval pH 7–8.
• 4. The rate of dissociation of oxygen from each hemoglobin is similar and is kinetically homogeneous with rate constants decreasing from 200–250/sec at pH 6.2 to about 25–26 at pH 7.7 with or without 1 mM ATP.
• 5. The CO combination reaction forHoplias hemoglobin is kinetically heterogeneous at all pH values and forHoplerythrinus hemoglobin below pH 7.5. The fast and slow phases each account for about half the observed reaction. The kinetic heterogeneity is maximal at low pH for both hemoglobins. The fast phase forHoplias hemoglobin is more than twice as fast as that forHoplerythrinus hemoglobins.

     

Separation and characterization of the hemoglobin components ofPterygoplichthys pardalis,the acaribodo

• 1. The four main hemoglobin components of the hemolysate ofPterygoplichthys pardalis have been isolated and characterized.
• 2. The functional properties investigated for the isolated components comprise the effect of pH and ATP on (i) the O₂ equilibrium, (ii) the O₂ dissociation kinetics, (iii) the CO combination kinetics.
• 3. Component I, corresponding to approx 50% of the total hemoglobin, is characterized by functional properties which are distinctly different from those of Components II, III and IV, which are alike
• 4. Thus it is shown, once more, that multiple components in an hemolysate fall into categories of hemoglobins characterized by distinct and complementary functional properties

     

Haemoglobin in male Daphnia magna

• 1.1. To define the respiratory function of haemoglobin in male Daphnia magna, the swimming activity, the depression of oxygen uptake by treatment with carbon monoxide and in vivo oxygenation of Hb at various oxygen pressures were investigated.
• 2.2. The P₅₀, values for the purified Hbs from male and female red animals were 2.0 and 2.7 torr in 0.1 M phosphate buffer (pH 7.2) at 20°C, respectively.
• 3.3. The isoelectric focusing patterns of the purified Hbs from male and female red animals showed only small differences in Hb components of high PI values.

     

Functional properties of hemoglobin and whole blood in an aquatic mammal,the Amazonian manatee (Trichechus inunguis)

• 1. Hematocrit (43%) and O₂ binding capacity (18.8 ml O₂/100 ml blood) ofTrichechus inunguis blood are low compared to the values for other diving mammals but are similar to those for land mammals.
• 2. Stripped manatee Hb is similar to human Hb A in its sensitivity to pH, 2,3-diphosphoglycerate and CO₂, but less sensitive to temperature and more prone to dissociate into dimers.
• 3. The unique Hill plots exhibit no cooperativity below 30% O₂-saturation indicating a highly stabilized T or low-affinity state(s); such asymmetric Hill plots together with biphasic O₂-binding kinetics could mean chain heterogeneity.
• 4. The pH dependence of oxygen binding by the apparent T state, hemoglobin as seen in the Hill plots, is enhanced by 2,3-diphosphoglycerate but eliminated by CO₂.