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.

     

Effects of erythrocytic nucleoside triphosphates on oxygen equilibria of composite and fractionated hemoglobins from the facultative air-breathing amazonian catfish,Hypostomus andPterygoplichthys

• 1. Oxygen equilibria ofHypostomus andPterygoplichthys hemoglobins and their sensitivities to the erythrocytic nucleotide triphosphates (NTP), ATP and guanosine triphosphate (GTP) are studied to investigate the mechanisms by which blood adapts to air- and water-breathing (cf. Weberet al., 1979).
• 2. Hemoglobins of both species are heterogeneous. All hemoglobin fractions isolated by iso-electric focusing reveal a high sensitivity to NTP, but GTP depresses O₂ affinity about twice as effectively as ATP. A cathodal hemoglobin component with a reversed Bohr effect was found inPterygoplichthys but not inHypostomus.
• 3. The data are discussed in relation to thein vivo cofactor modulation of blood O₂ affinity and the adaptive significance of functional heterogeneity of fish hemoglobins.

     

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 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.

     

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.

     

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.

     

Respiratory properties of blood and hemoglobin solutions from the piranha

• 1. Respiratory properties of piranha blood are distinguished from those of other fish primarily by the high CO₂ buffering capacity (ΔHCO₃/⁻ΔpH= 19.6mmol/l for oxygenated blood and 39.1 mmol/l for deoxygenated blood).
• 2. The concentration of nucleoside triphosphates (NTP) and the half-saturation tension (P₅₀) of whole blood were found to be inversely related to body size.
• 3. The higherP₅₀ in smaller fish, analogous to values obtained in previous studies involving interspecies comparisons, could be adaptive to a higher weight-specific metabolic rate.
• 4. Both ATP and guanosine triphosphate (GTP) lowered the oxygen affinity of purified hemoglobin solutions, accounting for the size-dependent correlation ofP₅₀ and NTP concentration in whole blood.
• 5. While similar in concentration in red cells, GTP is more potent than ATP as an allosteric modifier of hemoglobin function.

     

Equilibria and kinetic of oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish,Lepidosiren paradoxa

• 1. The haemoglobin of the South American lungfishLepidosiren paradoxa has a single component.
• 2. The equilibria of this respiratory protein with oxygen have been investigated both in the blood and with the purified haemoglobin. There is a substantial, normal, alkaline Bohr effect and marked sensitivity to organic phosphates in the haemoglobin solutions.
• 3. Studies on the pH dependence of the kinetics of oxygen dissociation can be interpreted in terms of a normal Bohr effect.
• 4. The kinetics of combination of carbon monoxide have an unusual pH dependence.
• 5. These findings are discussed in terms of the two-state model of Monodet al. (1965)

     

The effect of temperature on the oxygen equilibria of fish hemoglobins in relation to environmental thermal variability

• 1. We have measured the temperature dependence of the oxygen equilibria of blood and hemoglobin solutions from four neotropical and three temperate zone fishes.
• 2. Significant differences exist in the heats of oxygenation of the bloods but not the hemoglobin solutions from different species.
• 3. The differences in thermal sensitivity of the oxygenation of the bloods appear to depend on differences in intracellular pH, organic phosphates and other ligand-linked variables and not the intrinsic enthalpies of the oxygenation of the hemoglobins themselves.

     

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.

     

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 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.

     

Seasonal adaptations in oxygen transport in brown trout Salmo trutta fario

• 1.1. Blood parameters determining oxygen capacity and oxygen affinity were measured in brown trout at different times of the year.
• 2.2. Haematological data indicate a slight decrease in blood oxygen capacity during the warm seasons. 3. Oxygen affinity increases significantly during summer and decreases in winter.
• 3.4. The changes in P₅₀ exhibited a positive correlation with the amount of anodic haemoglobin components, and a negative correlation with the amount of cathodic haemoglobin components.
• 4.5. The changes observed in the [ATP]/[Hb] molar ratio were not correlated with oxygen affinity and gave values near one.
• 5.6. We conclude that the oxygen affinity increase could be a physiological adaptation to oxygen transport during the wanner period. A possible mechanism is discussed.

     

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.

     

Oxygen binding characteristics of Artemia hemoglobin domains

• 1.1. The extracellular hemoglobins of the crustacean Artemia can be split into structural and functional domains by limited proteolysis.
• 2.2. The oxygen affinity of the multi-domain fragments increases linearly with decreasing molecular weight.
• 3.3. Cooperativity is expressed only in the intact dimeric molecule and not at the subunit or multi-domain level.

     

Adaptative features of amazon fishes: Hemoglobins,hematology, intraerythrocytic phosphates and whole blood Bohr effect of Pterygoplichthys multiradiatus (Siluriformes)

• 1.1. Hemoglobin, hematological parameters, intraerythrocytic phosphates and whole blood Bohr effect of Pterygoplichthys multiradiatus, from the Amazon river, were studied in three different conditions: in their natural environment, acclimated to normoxia and acclimated hypoxia conditions.
• 2.2. Nine anodal hemoglobin fractions were detected on starch gel electrophoresis. No qualitative differences in the Hb electrophoretic patterns were detected in the three studied groups.
• 3.3. Hematocrit, hemoglobin concentration, MCV, MCHC and MCH were different among studied conditions.
• 4.4. GTP was almost absent in the blood of animals in natural conditions and acclimated to hypoxia, but was present at a concentration similar to ATP in normoxic acclimated animals.
• 5.5. There is a tendency for higher Hb-O₂ affinity for hypoxic acclimated/acclimatized animals.

     

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.

     

Patterns of hemoglobin production in the adult newt (Triturus cristatus) erythron following induction of anemia

• 1.1. Changes in the hemoglobins present in many vertebrates have been observed during development and during anemic episodes.
• 2.2. A change in the number of hemoglobins present and their relative amounts was observed when adult Triturus cristalus newts were made anemic by injection of acetylphenylhydrazine.
• 3.3. Hemoglobin IV, which is a minor hemoglobin in healthy adults, was found to be a major component during the subsequent erythropoietic response to hemolytic anemia.
• 4.4. No new hemoglobin not already present in the non-anemic state was detected during the response to induced anemia.

     

Acclimation to hypoxic water in facultative air-breathing fish: Blood oxygen affinity and allosteric effectors

• 1. Blood oxygen affinities, erythrocytic nucleoside triphosphate concentrations (NTP) and other hematological parameters were measured in facultative air-breathing fish from the Amazon after acclimation to well-aerated (“normoxic”) and hypoxic water (PO₂ = 125–135 and 20–25 mm, respectively).
• 2. In the armored catfishHypostomus sp. andPterygoplichthys sp., hypoxia induces intermittent surfacing to gulp air and results in lower NTP levels, chiefly through significant decreases in guanosine triphosphate (GTP). The subsequent increases in blood O₂ affinity appear adaptive to lowered time average internal O₂ tensions. No similar changes were seen in the ellSynbranchus which breathes air almost continuously when kept in hypoxic water.
• 3. The results are discussed in terms of their adaptive significance, and compared with data on temperate fish.

     

The hemoglobin ofpseudodoras,a South American catfish: Isolation,characterization and ligand binding studies

• 1. The hemoglobin of the Amazonian catfishPseudodoras sp. was isolated and characterized; it comprises a single component.
• 2. The hemoglobin's subunit composition is similar to that of other teleost hemoglobins. The apparent native molecular weight as determined by gel filtration is 66,000. The apparent subunit molecular weight is 14,300 by sodium dodecyl sulfate electrophoresis. The hemoglobin does not polymerize after oxidation by potassium ferricyanide.
• 3. The hemoglobin lacks a Root effect. A small Bohr effect is evident in the phosphate-free hemoglobin:Δlog p_1/2/ΔpH is no more than about −0.1 to −0.2 and increases toΔlog p_1/2/ΔpH = −0.4 in the presence of 1 mM ATP. The cooperativity, as determined byn of the Hill equation, is low, varying from 0.8 to 1.7 between pH 6.1 and 8.6.
• 4. Thep_1/2 values of stripped hemoglobin solutions are extremely low, less than 0.5 mm Hg at all pH values examined between pH 6.1 and 9.0. The high oxygen affinity is reflected primarily in the CO combination rate which resembles that found in myoglobins and isolated subunits of human hemoglobin.
• 5. Both the CO combination rate and the O₂ dissociation rate determined by stopped-flow spectrophotometry are pH and phosphate sensitive. Between pH 6.2 and 8.1 the CO_on rate increases about 5-fold in the phosphate-free hemoglobin. Addition of 1 mM ATP causes a depression in the rate at all pH values examined. The O_2off rate decreases 7-fold going from pH 6.0 to 8.2 in stripped hemoglobin solutions. Addition of 1 mM ATP induces a 10-fold decrease over the same range. At pH values below 6.0 a depression in the O_2off rate occurs in the stripped hemoglobin, indicative of an acid Bohr effect.