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.     

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).     

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.     

Hemoglobin and oxygen: different affinities in two species of rodents (Mus musculus and Pitymys duodecimcostatus)

Five different hemoglobins have been demonstrated by polyacrylamide-gel disk electrophoresis in the species Mus musculus. Oxygen affinities of hemoglobin (P50) from Mus musculus and Pitymys duodecimcostatus hemolysates were determined at pH 7.4 and 37 degrees C. Values obtained for delta log P50/delta pH in hemolysates from both species point out a more pronounced Bohr effect in Pitymys duodecimcostatus.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Temperature effects on hemocyanin oxygen binding in an antarctic cephalopod

The functional relevance of oxygen transport by hemocyanin of the Antarctic octopod Megaleledone senoi and of the eurythermal cuttlefish Sepia officinalis was analyzed by continuous and simultaneous recordings of changes in pH and hemocyanin oxygen saturation in whole blood at various temperatures. These data were compared to literature data on other temperate and cold-water cephalopods (octopods and giant squid). In S. officinalis, the oxygen affinity of hemocyanin changed at deltaP50/degrees C = 0.12 kPa (pH 7.4) with increasing temperatures; this is similar to observations in temperate octopods. In M. senoi, thermal sensitivity was much smaller (<0.01 kPa, pH 7.2). Furthermore, M. senoi hemocyanin displayed one of the highest levels of oxygen affinity (P50 < 1 kPa, pH 7.6, 0 degrees C) found so far in cephalopods and a rather low cooperativity (n50 = 1.4 at 0 degrees C). The pH sensitivity of oxygen binding (delta log P50/delta pH) increased with increasing temperature in both the cuttlefish and the Antarctic octopod. At low PO2 (1.0 kPa) and pH (7.2), the presence of a large venous oxygen reserve (43% saturation) insensitive to pH reflects reduced pH sensitivity and high oxygen affinity in M. senoi hemocyanin at 0 degrees C. In S. officinalis, this reserve was 19% at pH 7.4, 20 degrees C, and 1.7 kPa O2, a level still higher than in squid. These findings suggest that the lower metabolic rate of octopods and cuttlefish compared to squid is reflected in less pH-dependent oxygen transport. Results of the hemocyanin analysis for the Antarctic octopod were similar to those reported for Vampyroteuthis--an extremely high oxygen affinity supporting a very low metabolic rate. In contrast to findings in cold-adapted giant squid, the minimized thermal sensitivity of oxygen transport in Antarctic octopods will reduce metabolic scope and thereby contribute to their stenothermality.     

Oxygen transfer characteristics of the blood of reedfish, Erpetoichthys calabaricus

Oxygen transport characteristics and phosphate compounds were measured in the blood of reedfish, Erpetoichthys calabaricus, a bimodal breather. Blood from reedfish possessed the following values (mean +/- SD): hematocrit (21.7 +/- 0.4%), hemoglobin concentration (7.53 +/- 1.75 g%), red blood cell count (0.45 +/- 0.10 X 10(6)/mm3) and oxygen capacity (10.1 +/- 2.3 vol%). Although hematocrit, hemoglobin concentration, red blood cell count and oxygen capacity were all highly intercorrelated (P less than 0.01 in all cases), none of these parameters were significantly correlated with sex, weight or length in our sixteen fish sample. Erythrocyte volumes equalled 480 micrometers3, showed less variation (CV = 10.4%) and did not correlate with any other measured variable. Blood oxygen dissociation curves were sigmoidal and the P50's equalled 17.34 +/- 3.04 at 1% CO2 and 25 degrees C. Mean Bohr shift (delta log P50/delta pH) was -0.274 +/- 0.087. Temperature strongly influenced blood oxygen affinity. At 1% CO2, delta log P50/delta T equalled 0.026 +/- 0.006 (mean +/- SD). These hematological properties indicate that the blood of reedfish is similar to those of other tropical air-breathering species. Concentrations of total phosphate in the erythrocytes and percentage of total phosphate bound as nucleotide triphosphates were high. Surprisingly, 2,3diphosphoglycerate was found which has been reported in the erythrocytes of only two other fish species. Blood characteristics of reedfish exposed to air for 4 hr with one exception (Hill numbers) were not significantly different from water exposed controls. This suggests that the reedfish does not possess blood respiratory mechanisms to facilitate respiration solely by air-breathing.     

Gas transfer system in Alvinella pompejana (Annelida polychaeta, Terebellida): functional properties of intracellular and extracellular hemoglobins

Alvinella pompejana is a tubicolous polychaete that dwells in the hottest part of the hydrothermal vent ecosystem in a highly variable mixture of vent (350 degrees C, anoxic, CO(2)- and sulfide-rich) and deep-sea (2 degrees C, mildly hypoxic) waters. This species has developed distinct-and specifically respiratory-adaptations to this challenging environment. An internal gas exchange system has recently been described, along with the report of an intracellular coelomic hemoglobin, in addition to the previously known extracellular vascular hemoglobin. This article reports the structure of coelomic hemoglobin and the functional properties of both hemoglobins in order to assess possible oxygen transfer. Coelomocytes contain a unique monomeric hemoglobin with a molecular weight of 14,810+/-1.5 Da, as determined by mass spectrometry. The functional properties of both hemoglobins are unexpectedly very similar under the same conditions of pH (6.1-8.2) and temperature (10 degrees -40 degrees C). The oxygen affinity of both proteins is relatively high (P50=0.66 Torr at 20 degrees C and pH 7), which facilitates oxygen uptake from the hypoxic environment. A strong Bohr effect (Phi ranging from -0.8 to -1.0) allows the release of oxygen to acidic tissues. Such similar properties imply a possible bidirectional transfer of oxygen between the two hemoglobins in the perioesophagal pouch, a mechanism that could moderate environmental variations of oxygen concentration and maintain brain oxygenation.     

Red blood cell function and haematology in two tropical freshwater fishes from Australia

Salmon catfish and tarpon occur in habitats that periodically become deficient in oxygen resulting in high mortalities of other fish species. The water-breathing catfish, Arius leptaspis, and the facultative air-breathing tarpon, Megalops cyprinoides, both have high haemoglobin and haematocrit, and the oxygen carrying capacity in the air-breather is exceptionally high (15.6+/-1.2 vol%). Iso-pH oxygen equilibria of the red blood cells at 25 degrees C revealed high affinity (P(50)=9 mmHg, pH 7.4) and co-operativity (n(50)>2.2, pH 7.4) in the catfish, and contrasted with low affinity (P(50)=32 mmHg, pH 7.4) and co-operativity (n(50) approximately 1) in the air-breathing tarpon. Oxygen binding was further distinguished by relative pH insensitivity (Bohr factor, ?=Deltalog P(50)/Deltalog pH=-0.22) in the catfish, compared with a significant Bohr effect in the tarpon (?=-0.96). The potential for modulation of haemoglobin-oxygen affinity was indicated by a high ratio of GTP to ATP in the erythrocytes of the catfish, whereas regulation in the tarpon appeared due to ATP alone. Differences in blood respiratory functions between the two species are likely to reflect reduced opportunity for activity under extreme hypoxia in the catfish.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

Functional and biochemical properties of the hemoglobins of the burrowing brittle star Hemipholis elongata say (Echinodermata,Ophiuroidea)

The burrowing brittle star Hemipholis elongata (Say) possesses hemoglobin-containing coelomocytes (RBCs) in its water vascular system. The RBCs, which circulate between the arms and body, are thought to play a role in oxygen transport. The hemoglobin of adult animals has a moderate affinity for oxygen (P(50) = 11.4 mm Hg at pH 8, 20 degrees C, measured in cellulo) and exhibits cooperativity (Hill coefficient > 1.7). The hemoglobin of juveniles has a higher affinity (P(50) = 2.3 mmHg at pH 8.0, 20 degrees C) and also exhibits cooperativity. The oxygen-binding properties of the hemoglobin are relatively insensitive to pH, temperature, and hydrogen sulfide. Adult hemoglobin is a heterogeneous mixture composed of three major fractions. The combined results of electrospray mass spectrometry and oxygen-binding experiments performed on purified fractions indicate that the native hemoglobin is in the form of homopolymers. A partial amino acid sequence (about 40 amino acids) of adult hemoglobin reveals little homology with holothurian hemoglobins.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

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.     

Hemoglobin-oxygen-affinity and acid-base properties of blood from the fossorial mole-rat, Cryptomys hottentotus pretoriae

Oxygen affinity and other hematological parameters in strictly subterranean mole-rats, Cryptomys hottentotus (subspecies pretoriae) were measured immediately upon capture and after 14-21 days in captivity. The pH, hematocrit, hemoglobin (Hb) concentration, blood oxygen content, 2,3 bisphosphoglycerate (2,3 BPG) concentration and oxygen dissociation curves (ODC), as well as tonometric measurements, were determined using whole blood. Additionally ODCs were also determined for stripped hemolysates of individual animals. Compared to other mammals, blood of freshly caught animals had low pH (7.32+/-0.22), elevated hematocrits (48.4+/-3.8 %) and significantly lower P50 values for whole blood (21.1+/-1.6 mm Hg at pH 7.4) than those reported for other similar-sized fossorial and terrestrial mammals. Blood carbon dioxide content (22.4+/-3.9 mMol L(-1)), hemoglobin concentration (1.9+/-0.15 mMol L(-1)), oxygen content (164.8+/-26 mL L(-1)), bicarbonate concentrations (22.5+/-3.5 mMol L(-1)) were within the range of values reported for similar-sized mammals. We conclude that high blood-oxygen affinity, low body temperature and possibly also high hematocrit enable C. h. pretoriae to maintain an adequate oxygen supply to the tissues in a potentially hypoxic burrow atmospheres, but that the blood of this species shows no exceptional CO2 sensitivity or buffering capacity.     

Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant.

Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.     

Thermal stability and functional properties of human hemoglobin in the presence of aliphatic alcohols]

Differential scanning microcalorimetry was used to study thermal stability of the ferro- and ferriforms of hemoglobin at pH 7.4 in phosphate buffer and in buffer mixtures of methanol, ethanol, 1-propanol. Denaturation of the human hemoglobin molecule composed of four subunits was cooperative transition. The thermostability of the hemoglobin forms decreased in the order of carboxyhemoglobin (TD = 82.0 degrees C) > oxyhemoglobin (71.0 degrees C) > methemoglobin (67.0 degrees C). The aliphatic alcohols as cosolvents decreased the hemoglobin stability because of loosening the structure of the globin moiety by disturbing its hydrophobic contacts and hydrogen bonds. These alcohols reduced the oxygen affinity for hemoglobin probably due to perturbation of the R<-->T equilibrium by the decreased bulk dielectric constant of the solvent. Oxyhemoglobin and methemoglobin was converted to hemichrome by high alcohol concentrations.     

Cyanate binding to the ferric heme octapeptide from cytochrome c. A model for anion binding to high spin ferric hemoproteins

Equilibrium constants for the binding of cyanate to the ferric heme c octapeptide in 50% ethylene glycol, 50% aqueous buffer were measured spectrophotometrically. Equilibrium constants measured at several temperatures from -20 degrees C to 0 degrees C exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta Ho = -1.3 X 10(3) +/- 0.9 X 10(3) J/mol (-3.1 X 10(2) +/- 2 X 10(2) cal/mol), delta So = -3 +/- 3 J/K X mol (-0.6 +/- 0.8 cal/K X mol). The equilibrium constant for cyanate binding at 25 degrees C and pH 7.4 is 1.21 which is approximately 2 to 3 orders of magnitude lower than that observed for cyanate binding to methemoglobin and metmyoglobin. Krel, the ratio of the hemoprotein to model heme octapeptide binding constants, for NCO- is smaller than Krel for N3- suggesting that hydrogen bonding between the terminal ligand atoms and the distal histidine in hemoglobin and myoglobin does not contribute to the increased protein ligand stabilization observed for these anions relative to the model. A donor-acceptor interaction between the distal histidine and the electrophilic middle atoms of these bound ligands is proposed.     

Characterization of the hemoglobins of the Australian lungfish Neoceratodus forsteri (Krefft)

We examined for the first time the hemoglobin components of the blood of the Australian lungfish, Neoceratodus forsteri and their functional responses to pH and the allosteric modulators adenosine triphosphate (ATP), guanosine triphosphate (GTP), 2,3-bisphosphoglyceric acid (BPG) and inositol hexaphosphate (IHP) at 25 degrees C. Lysates prepared from stripped, unfractionated hemolysate produced sigmoidal oxygen equilibrium curves with high oxygen affinity (oxygen partial pressure required for 50% hemoglobin saturation, p(50)=5.3 mmHg) and a Hill coefficient of 1.9 at pH 7.5. p(50) was 8.3 and 4.5 mmHg at pH 6 and 8, respectively, which corresponded to a modest Bohr coefficient (Delta log p(50)/Delta pH) of -0.13. GTP increased the pH sensitivity of oxygen binding more than ATP, such that the Bohr coefficient was -0.77 in the presence of 2 mmol L(-1) GTP. GTP was the most potent regulator of hemoglobin affinity, with concentrations of 5 mmol L(-1) causing an increase in p(50) from 5 to 19 mm Hg at pH 7.5, while the order of potency of the other phosphates was IHP>ATP>BPG. Three hemoglobin isoforms were present and each contained both alpha and beta chains with distinct molecular weights. Oxygen affinity and pH-dependence of isoforms I and II were essentially identical, while isoform III had a lower affinity and increased pH-dependence. The functional properties of the hemoglobin system of Neoceratodus appeared consistent with an active aquatic breather adapted for periodic hypoxic episodes.     

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.