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.

     

Comparative study of the oxyhaemoglobin dissociation curve of four mammals: Man,dog, horse and cattle

• 1.1. The entire oxygen dissociation curve (ODC) and the effects of temperature, pH and 2,3-diphosphoglycerate (DPG) on this curve, have been compared in four mammalians: man, dog, horse and cattle.
• 2.2. If the oxyphoric capacities are similar between these species (around 1.39ml O₂/gHb), their P50, measured in standard conditions, i.e. at pH 7.4;.pCO₂ 40mmHg and T 37°C, varies between 23.8 (± 0.8) mmHg for the horse, 25.0 (± 1.4) mmHg for cattle, 26.6 (± 1.2) for man and 28.8 (± 2.6) mmHg for the dog.
• 3.3. The higher dispersion of the dog's P50 is due to difference between breeds; in seven breeds investigated, the P50 ranges from 25.8 (spaniel) to 35.8 (hound).
• 4.4. We noted no sex difference in the four species.
• 5.5. The DPG level is confirmed to be low in cattle (< 1 μmol/gHb) as compared to man (13.5 ± 2.1 gmmol/gHb), horse (16.9 ± 1.1 gmmol/gHb) and dog (19.4 ± 2.8 μmol/gHb).
• 6.6. The oxygen exchange fraction defined as the difference in vol% between a pO₂ of 80 and 35 mmHg is, respectively, 3.6 (± 0.6) vol% for cattle, 4.0 (0.4) vol% for the horse, 5.5 (± 0.5) vol% for man and 6.6 (± 1.7) vol% for the dog.
• 7.7. The position and shape of the ODC, as well as T, DPG and pH effects, indicate that the haemoglobin of man and dog seem better adapted to O₂ delivery as compared to the horse and cattle.

     

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.

     

O2-binding by the blood of the landhopper,Arcitalitrus dorrieni (Hunt) (Crustacea: Amphipoda)

• 1.1. The O₂-binding characteristics of the blood of the euterrestrial amphipod (landhopper) Arcitalitrus dorrieni have been studied.
• 2.2. The blood exhibited a low O₂ affinity, with a p₅₀ (at pH = 7.8) of 21.4 torr (10°C). Affinity decreased with an increase in temperature at constant pH (ΔH = − 79.4kJ/mol) but the Bohr factor (ΔlogP₅₀/Δ pH = −0.67) was unaffected.
• 3.3. The O₂-carrying capacity of the blood was moderate (1.51 ml/100 ml)
• 4.4. The results support the hypothesis that the blood of terrestrial amphipods is characterized by having a low affinity pigment.

     

The effect of d2o on glycolysis by rat hepatocytes

• 1.1. The effect of incorporating D₂O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined.
• 2.2. The substitution by heavy water, D₂O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO₂ yields from glucose. At 10 mM glucose, 40% D₂O doubled glucose uptake, increased CO₂ production by 40%, and increased lactate production by 350%.
• 3.3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose.
• 4.4. There was no effect on CO₂ production above glucose concentrations of 30 mM.
• 5.5. Ten percent D₂O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D₂O the inhibition ranged from 10 to 20%.
• 6.6. No effect of D₂O on the rate of glucokinase or glucose-6-phosphatase was observed.
• 7.7. The concentration of fructose, 2,6-P was not affected by D₂O

     

Effects of exercise-stress on ventilation,cardiac output and blood respiratory parameters in the carp,Cyprinus carpio

• 1.1. Carp (Cyprinus carpio) were stressed to exercise by rolling in a respiration chamber. Ventilatory water flow rate, cardiac output and blood respiratory parameters were determined.
• 2.2. During exercise, oxygen uptake increased about 3.5-fold and returned to pre-exercise level within 15 min.
• 3.3. This exercise-stress resulted in no plasma acidosis and in no swelling of the erythrocytes.
• 4.4. Ventilatory water flow rate increased 6-fold, whereas cardiac output increased 2-fold. Hence the ventilation-perfusion ratio increased during exercise.
• 5.5. During exercise, arterial O₂ content (CaO₂) increased due to increases in O₂ tension (PaO₂), O₂ saturation of hemoglobin (SaO₂) and hemoglobin concentration (Hb). On the other hand, Pv̄O₂ and Sv̄O₂ remained at the resting levels but Cv̄O₂ slightly increased due to an increase in Hb.
• 6.6. Arterial-venous O₂ difference (CaO₂-Cv̄O₂) increased by 38%, which was met by a much greater increase in CaO₂ than Cv̄O₂.

     

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.

     

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.

     

Aerobic and anaerobic respiration of adult Artemia salina L., acclimated to different oxygen concentrations

• 1.1. Adult male Atremia salina L. were acclimated to five different oxygen concentrations and their respiration in response to environmental oxygen concentrations was determined.
• 2.2. Anemia is a respiratory regulator over a wide range of partial O₂ pressures. A critical oxygen tension exists and decreases with acclimation to lower pO₂.
• 3.3. Hypoxic conditions induce the production of hemoglobin III.
• 4.4. Lactic acid is produced during anaerobiosis.
• 5.5. Production of Hb III and lactic acid, being inversely proportional to the acclimation level, has to be considered as a long term or short term adaptation to hypoxic conditions.

     

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

     

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

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.

     

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.

     

Comparative study of hemoglobins from different Artemia populations. The influence of temperature on the oxygen equilibria

• 1.1. The P50 values of extracellular hemoglobin (Hb) of five Artemia populations from different geographical origin are affected by temperature.
• 2.2. The free oxygen binding energy is high for all the populations (ΔH between −34.7 and −56.2kj/mol).
• 3.3. A possible correlation between thermal sensitivity of Hb and the ambient temperature of the habitat must be considered very carefully.
• 4.4. The occurence of different quantities of Hb1 (αα chains) Hb2 (αβ chains) and Hb3 (ββ chains) in the different populations possibly influences thermal sensitivity.

     

Effect of pH on metabolism of the glutamine carbon skeleton by renal cortical mitochondria

• 1.1. To determine the effect of altered acid-base homeostasis on the intramitochondrial metabolism of the glutamine carbon skeleton ¹⁴CO₂ production from [U-¹⁴C]glutamine by isolated rat renal cortical mitochondria was measured.
• 2.2. Mitochondria from rats with chronic metabolic acidosis either showed no change or diminished ¹⁴CO₂ production in comparison with pair fed controls.
• 3.3. By contrast, when the pH of the medium incubating mitochondria from normal rats was manipulated (pH 7.0, 7.4, 7.7), ¹⁴CO₂ production was clearly altered, but the direction and magnitude of the change depended on the glutamine concentration used (0.5 or 10.0 mM).
• 4.4. Mitochondria produced significant quantities of ¹⁴CO₂ when [1,4 ¹⁴C]succinate was used as substrate, indicating that ¹⁴CO₂ production from glutamine does not originate solely from the decarboxylation of α KG.
• 5.5. Thus chronic acidosis and pH, per se, affect intramitochondrial glutamine carbon skeleton metabolism in different fashions, but the specific mechanism cannot be elucidated using ¹⁴CO₂ production from [U-¹⁴C]glutamine.
• 6.6. Additional studies directly quantitating the metabolic products of glutamine have confirmed these findings and more precisely defined the sites of metabolic alteration.

     

The effects of extracellular-and intracellular-pH on transmitter mobilization and fractional release in Helix pomatia

• 1.1. Transmitter mobilization and fractional release were studied in Helix pomatia. The right palliai nerve was stimulated and a synaptic potential was recorded in cell F76 in the right parietal ganglion.
• 2.2. The extra- and intra-cellular pH were changed with Tris-maleate, CO₂ or (NH₄)₂SO₄.
• 3.3. The time constant for the monoexponential part of mobilization decreased with reduced intracellular pH. Only a fraction of this effect could be related to an increase in the intracellular Ca-activity.
• 4.4. Fractional release was reduced in low external pH, but was increased in low intracellular pH. Fractional release is affected more by changes in internal pH than external pH.

     

Gas tensions within the egg of the quail (Coturnix c. Japonica) during the closing stages of embryonic development

• 1.1. The respiratory status of the embryonic quail during the two days prior to hatching was assessed by measuring the gas tensions within the air space of the egg and of blood collected from the chorioallantois.
• 2.2. When the lungs became inflated there was a significant decrease in the pO₂ of the gas in the air space.
• 3.3. After pipping, there was a rise in the pO₂ and fall in the pCO₂ within the air space, together with corresponding changes in the blood.
• 4.4. The outer shell membrane remained intact until the onset of hatching.
• 5.5. These results were compared with those obtained by other workers using the domestic fowl.

     

Oxygen consumption and haemocyanin function in the freshwater snail Marisa cornuarietis (L.)

• 1.1. The MO₂ for branchial respiration in adult snails increased from 0.24 mmol/l/O₂ kg/hr at 18°C to 0.83 mmol/l/O₂ kg/hr at 40°C. Q₁₀ values were 2.75 between 35 and 40°C and 1.8 between 18 and 30°C.
• 2.2. The haemocyanin (31.9 ± 5.8 mg/ml) has a high oxygen affinity (6.28 ± 0.8 at 25°C) with a reversed Bohr effect measured between a pH of 6.80 and 7.95 with gelchromatographed haemolymph, and measured between a pH of 7.34 and 8.10 for native haemolymph.
• 3.3. Growth rate is optimal between 27 and 30°C whilst at 24°C stunted growth was found.
• 4.4. At 25°C the same MO₂ values were found for aerial and aquatic respiration.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

The effect of temperature on the acid-base status of the blood of the hatching quail (Coturnix c. japonica)

• 1.1. The ambient temperature of embryos of pipped eggs was reduced from 38 to 28°C for a period of 45 min.
• 2.2. The blood P_CO2 was lower and the blood more alkaline at 28°C than at 38°C.
• 3.3. At 28°C plasma [HCO₃⁻] ] was lower than predicted from the blood buffer line determined in vitro.
• 4.4. The plasma concentrations of strong ions and lactate were the same at both temperatures.
• 5.5. After the ambient temperature had been returned to 38°C for a period of 45 min, blood pH was more acidic than before cooling, but there was no difference in blood P_CO2.
• 6.6. The plasma [HCO₃⁻] was the same as that at 28°C and plasma [K⁺] was higher than before cooling.
• 7.7. The results arc discussed in relation to the factors affecting blood pH in embryos at this stage of development.