Temperature dependence of haemoglobin-oxygen affinity in heterothermic vertebrates: mechanisms and biological significance.

As demonstrated by August Krogh et al. a century ago, the oxygen-binding reaction of vertebrate haemoglobin is cooperative (described by sigmoid O(2) equilibrium curves) and modulated by CO(2) and protons (lowered pH) that - in conjunction with later discovered allosteric effectors (chloride, lactate and organic phosphate anions) - enhance O(2) unloading from blood in relatively acidic and oxygen-poor tissues. Based on the exothermic nature of the oxygenation of the haem groups, haemoglobin-O(2) affinity also decreases with rising temperature. This thermal sensitivity favours oxygen unloading in warm working muscles, but may become detrimental in regionally heterothermic animals, for example in cold-tolerant birds and mammals and warm-bodied fish, where it may perturb the balance between O(2) unloading and O(2) requirement in organs with substantially different temperatures than at the respiratory organs and thus commonly is reduced or obliterated. Given that the oxygenation of haemoglobin is linked with the endothermic release of allosteric effectors, increased effector interaction is an effective strategy that is widely exploited to achieve adaptive reductions in the temperature dependence of blood-O(2) affinity. The molecular mechanisms implicated in heterothermic vertebrates from different taxonomic groups reveal remarkable variability, both as regards the effectors implicated (protons in tunas, organic phosphates in sharks and billfish, chloride ions in ruminants and chloride and phosphate anions in the extinct woolly mammoth, etc.) and binding sites for the same effectors, indicating multiple evolutionary origins, but convergent physiological functionality (reductions in temperature dependence of O(2) -binding affinity that safeguard tissue O(2) supply).

Lacking deoxygenation-linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells.

AIM: Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. METHODS: The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3 protein, on the oxygenation reaction of HbF and HbA, isolated from umbilical chord red cells. RESULTS: Contrasting with the unequivocal interaction of the peptide with HbA that with fetal HbF is weak, and annihilated in the presence of autochthonous red cell O2 affinity modulators (chloride and organic phosphates). CONCLUSION: The data indicate that HbF does not function as a transducer mediating O2 dependence of glycolysis in fetal red cells, in accordance with the different O2 and metabolic profiles compared to those in HbA-bearing adult red cells. In conjunction with the previously discovered O2 dependence of K+ transport in HbF-rich fetal cells, they moreover argue against linkage between different, physiologically relevant, O2-dependent red cell functions.

Multiple strategies of Lake Victoria cichlids to cope with lifelong hypoxia include hemoglobin switching.

Many fish species adapt to hypoxia by reducing their metabolic rate and increasing hemoglobin-oxygen (Hb-O(2)) affinity. Pilot studies with young broods of cichlids showed that the young could survive severe hypoxia in contrast with the adults. It was therefore hypothesized that early exposure results in improved oxygen transport. This hypothesis was tested using split brood experiments. Broods of Astatoreochromis alluaudi, Haplochromis ishmaeli, and a tilapia hybrid (Oreochromis) were raised either under normoxia (NR; 80-90% air saturation) or hypoxia (HR; 10% air saturation). The activity of the mitochondrial citrate synthase was not different between NR and HR tilapia, but was significantly decreased in HR A. alluaudi and H. ishmaeli, indicating lowered maximum aerobic capacities. On the other hand, hemoglobin and hematocrit levels were significantly higher in all HR fish of the three species, reflecting a physiological adaptation to safeguard oxygen transport capacity. In HR tilapia, intraerythrocytic GTP levels were decreased, suggesting an adaptive increase of blood-O(2) affinity. Similar changes were not found in HR H. ishmaeli. In this species, however, all HR specimens exhibited a distinctly different iso-Hb pattern compared with their NR siblings, which correlated with a higher intrinsic Hb-O(2) affinity in the former. All HR cichlids thus reveal left-shifted Hb-O(2) equilibrium curves, mediated by either decreased allosteric interaction or, in H. ishmaeli, by the production of new hemoglobins. It is concluded that the adaptation to lifelong hypoxia is mainly due to improved oxygen transport.

Effects of short-term hypoxia on neuroglobin levels and localization in mouse brain tissues.

Nerve cells are highly susceptible to ischemic and hypoxic injuries. The neuroglobin (Ngb), found in vertebrate nerve cells, has been suggested to protect nerve cells from ischemic episodes by a yet unknown mechanism. However, contradicting reports exist regarding localization and up-regulation of Ngb in response to hypoxia. The aim of the present study was to probe the distribution of Ngb proteins in mouse brain and retina by immunohistochemistry, and to quantify the levels of Ngb mRNA by reverse-transcription-polymerase chain reaction (RT-PCR) after short-term (2 h) exposure to 7.6% oxygen. We found Ngb to be present throughout the neocortex, most abundantly in the perirhinal, entorhinal and temporal cortical areas, the thalamus and hypothalamus, the choroid plexus, the olfactory bulb and the cranial nerve nuclei in the brainstem. Intense staining was observed in the mesencephalic central grey area and the Purkinje cells. Two-hour hypoxic exposure caused no detectable changes in staining intensity or spatial distribution of Ngb neither in the Purkinje cells nor in any other brain areas observed. The RT-PCR data supported the lack of differences in brain Ngb levels between normal and oxygen-deprived animals. In the retina, Ngb localization by immunohistochemistry was confined to the inner segments of the photoreceptors, the plexiform layers and the ganglion cells. Short-termed hypoxia did not change retinal Ngb levels as assessed by both techniques. The lack of Ngb up-regulation in the brain is consistent with results from previous long-term hypoxic experiments, suggesting that Ngb is not regulated by pure hypoxia in vivo.

New insights into the proton-dependent oxygen affinity of Root effect haemoglobins.

A long-standing puzzle with regard to protein structure/function relationships is the proton-dependent modification of haemoglobin (Hb) structure that causes oxygen to be unloaded from Root effect Hbs into the swim bladders and eyes of fish even against high oxygen pressure gradients. Although oxygen unloading in Root effect Hbs has generally been attributed to proton-dependent stabilization of the T-state, protonation of Root effect Hbs can alter their ligand affinities in both R- and T-state conformations and either stabilize the T-state or destabilize the R-state. The C-terminal residues that are so important in the Bohr effect of human Hb appear to be involved in the Root effects of some fish Hbs and not in others, indicating that several evolutionary pathways have resulted in expression of highly pH-dependent Hbs. New data are presented that show surprising similarities in the pH- and anion-dependence of sulfhydryl group reactivity and anaerobic oxidation of human and fish Hbs. The available evidence supports the concept that in both Bohr effect and Root effect Hbs a large steric component acts in addition to quaternary shifts between R and T conformations to regulate ligand affinity. Allosteric effectors moderate these steric effects within both R- and T-state conformations and allow for an elegant match between Hb function and the wide-ranging physiological needs of diverse organisms.

Comparison of stone size and response to analgesic treatment in predicting outcome of patients with renal colic.

The aim of this study was to compare the prognostic value of stone size and response to analgesic treatment in patients with renal colic. We reviewed the charts of patients treated for renal colic in our Emergency Department. The eligibility criteria were a radiological examination demonstrating direct or indirect signs of ureteral obstruction and/or a stone. The primary endpoint was the requirement for surgical treatment. The parameters considered as prognostic factors were pain relief with ketorolac (K) or ketorolac plus opiate treatment (KO), and stone size (>or= or <6 mm). Ninety-five patients were considered for analysis. Of these, 49 (52%) had a stone demonstrated radiologically. Four out of 27 patients (15%) in the KO group and six out of 68 patients (8.8%) in the K group required a surgical procedure to relieve the obstruction (NS). Four out of five patients (80%) with a stone >or=6 mm required a surgical procedure, compared with one out of 44 (2.2%) who had a stone smaller than 6 mm (P<0.001). In conclusion, stone size is a better prognostic factor than the response to analgesic treatment in predicting the clinical outcome of patients with renal colic. A stone >or=6 mm in patients with renal colic should alert the emergency physician that urological complications requiring surgical intervention may occur and that urological management may be warranted.

Evaluation of emergency air evacuation of critically ill patients from cruise ships.

BACKGROUND: The study objectives were to assess the ship physician's diagnostic accuracy in making the decision to air evacuate critically ill patients from cruise ships, to determine the outcome of these patients, and the overall benefit of air evacuation. METHODS: From October 1999 to May 2000, we performed a prospective study of critically ill patients coming from cruise ships in the Caribbean and transported to our institution by air ambulance. Demographics, initial diagnosis, and treatment on board were collected by the triage officer at the time of the cruise physician's first call. In route complications and flight team composition were obtained from the air ambulance monitoring log. Patients were followed-up in the hospital for complications, outcome, and final diagnosis. RESULTS: A consecutive series of 104 patients were considered for analysis. There were 65 men and 39 women (mean age: 68.7 years). Cruise physician's diagnosis was correct in more than 90% of the cases. Internal medicine and surgical conditions represented 80.8% and 19.2% of the cases respectively, falling mainly into three categories: cardiac (34.6%), neurological (20.2%), and digestive (14%). Two cardiac arrests and 1 ventricular fibrillation were successfully resuscitated and 5 of 15 myocardial infarctions received thrombolytic therapy on board. Air transfers were warranted in 96.1% of the cases and physician presence in the flight was considered appropriate in 97.6%. In route complications and mortality rate were 5.8% and 2.9% respectively, related to serious cardiac events. Among the 98 hospitalized patients, 10 patients developed new complications and 5 died. The overall mortality rate was 7.7%. CONCLUSION: The cruise industry appears off to a good start in the medical treatment of passengers needing air evacuation to a land based medical facility. There is room for improvement and adoption of American College of Emergency Physicians (ACEP) and International Council of Cruise Lines (ICCL) Health Care Guidelines are meaningful first steps. Analysis of Caribbean medical facilities and implementation of active telemedicine conferencing represent alternatives to air evacuation that need to be studied.

Oxygen binding by single red blood cells from the red-eared turtle Trachemys scripta.

Oxygen-binding properties of single red blood cells from the red-eared turtle Trachemys scripta were measured by microspectrophotometry to describe the variation in oxygen affinity of red blood cells and to gain insight into the distribution of functionally different hemoglobins among red blood cells. Methodologically, this study represents the first report on the cell-to-cell variation in oxygen-binding properties based on oxygen-binding curves of single vertebrate red blood cells. The cells differed significantly with respect to oxygen affinity. Mean oxygen pressure at half saturation of the cells in a blood sample was found to be 20.1 +/- 3.3 (SD) Torr. The distribution of oxygen affinities among red blood cells is unimodal, indicating that the two hemoglobins found in turtle blood are not segregated in distinct cells. Therefore, the functional interaction shown by these hemoglobins in vitro is likely to take place in vivo. The considerable variation in oxygen affinity between individual red blood cells calls for its incorporation in models of tissue oxygenation.

Nonvertebrate hemoglobins: functions and molecular adaptations.

Hemoglobin (Hb) occurs in all the kingdoms of living organisms. Its distribution is episodic among the nonvertebrate groups in contrast to vertebrates. Nonvertebrate Hbs range from single-chain globins found in bacteria, algae, protozoa, and plants to large, multisubunit, multidomain Hbs found in nematodes, molluscs and crustaceans, and the giant annelid and vestimentiferan Hbs comprised of globin and nonglobin subunits. Chimeric hemoglobins have been found recently in bacteria and fungi. Hb occurs intracellularly in specific tissues and in circulating red blood cells (RBCs) and freely dissolved in various body fluids. In addition to transporting and storing O(2) and facilitating its diffusion, several novel Hb functions have emerged, including control of nitric oxide (NO) levels in microorganisms, use of NO to control the level of O(2) in nematodes, binding and transport of sulfide in endosymbiont-harboring species and protection against sulfide, scavenging of O(2 )in symbiotic leguminous plants, O(2 )sensing in bacteria and archaebacteria, and dehaloperoxidase activity useful in detoxification of chlorinated materials. This review focuses on the extensive variation in the functional properties of nonvertebrate Hbs, their O(2 )binding affinities, their homotropic interactions (cooperativity), and the sensitivities of these parameters to temperature and heterotropic effectors such as protons and cations. Whenever possible, it attempts to relate the ligand binding properties to the known molecular structures. The divergent and convergent evolutionary trends evident in the structures and functions of nonvertebrate Hbs appear to be adaptive in extending the inhabitable environment available to Hb-containing organisms.

Hemoglobin from a deep-sea hydrothermal-vent copepod.

Deep-sea hydrothermal-vent fauna live in a highly variable environment where oxygen levels can be very low, and carbon dioxide and sulfide can reach high concentrations (1). These conditions are harsh for most aerobic metazoans, yet copepods can be abundant at hydrothermal vents. Here we report the structure and functional properties of hemoglobin extracted from the copepod Benthoxynus spiculifer, which was found in large numbers in a paralvinellid/gastropod community collection made during a cruise to the Juan de Fuca Ridge in 1998. Although hemoglobin has been reported in some littoral copepods (2), this is the first study of the structure and functional properties of copepod hemoglobin. Hemoglobin represents about 60% of the total soluble proteins extracted from B. spiculifer, and although it imparts a red color to the copepod, it does not provide a significant storage pool of oxygen. It is a 208-kDa protein, composed of 14 globin chains--7 of 14.3 kDa and 7 of 15.2 kDa. The hemoglobin has a very high and temperature-sensitive oxygen affinity, with no cooperativity or Bohr effect. These properties are adaptive for an animal living in a low-oxygen environment in which the primary function of the hemoglobin is most likely oxygen acquisition to support aerobic respiration.

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

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.

Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale.

The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglycerate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O(2) affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (Hb(An) and Hb(Ca)) were isolated for functional and molecular characterization. In contrast to Hb(An), phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O(2) affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences of K(T) (O(2) association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. Hb(Ca)-O(2) equilibria analyzed in terms of the Monod-Wyman-Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation-linked binding to Hb(Ca), suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the alpha and beta chains of Hb(Ca) obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities.

The role of amino acid alpha38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland.

The role of the amino acid at position alpha(38) in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position alpha(38) (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln(38) of the human embryonic haemoglobin zeta-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of alphaGln(38) and betaTyr(145) is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the zeta-chain containing human embryonic haemoglobins.

The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from Lumbricus terrestris.

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to /=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.

Functional differentiation in trematode hemoglobin isoforms.

The Hbs and the major electrophoretic Hb components (isoHbs) were isolated from three species of the trematodes, Explanatum explanatum (Ee), Gastrothylax crumenifer (Gc) and Paramphistomum epiclitum (Pe), that parasitise the common Indian water buffalo Bubalus bubalis. The Hbs are monomeric and resemble the so-called nonfunctional mutant hemoglobins that have Tyr at B10 or E7 positions (replacing Leu and the His residues, respectively). However, they are capable of binding with O2 and CO. O2 equilibrium studies of trematode Hb isoforms reveal extremely high O2 affinities, with half-saturation O2 tension (P50) values up to 800 times lower than those of human hemoglobins. This correlates with Tyr residues at B10 and at the distal position (E7) that decrease the O2 dissociation rate by contributing hydrogen bonds (H-bonds) to the bound O2. These substitutions also increase the O2 association rates either due to orientation of E7-Tyr towards the solvent and/or by sterically hindering the entry of water molecules into the heme pocket. The latter may account for the low rate of autoxidation of trematode Hbs. The Hbs and their isoforms from different species exhibited pronounced variation in O2 affinity, which may relate to subtle differences in the structure of the heme pocket. The O2 affinities of the composite (unfractionated) Hbs were intermediate to those of the individual Hb isoform. The P50 values of Hbs here obtained by direct O2 equilibrium measurements differed from those calculated from kinetic data already published [Kiger, L., Rashid, A. K., Griffon, N., Haque, M., Moens, L.,Gibson, Q. H., Poyart, C., & Marden, M. C. (1998). Biophys. J. 75, 990-998.] Intermediate state(s) due to slow reorientation of E7-Tyr may account for this difference. Some Hb isoforms showed slight (either normal or reverse) Bohr effects. The hyperbolic O2 equilibrium curve, Hill coefficient (n) values near unity accord with a monomeric nature of trematode Hbs. In marked contrast to vertebrate Hbs, CO does not seem to compete effectively with O2 in trematode Hbs, as evident from partition coefficient values (M) below 1.

Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea, hydrothermal vent scaleworm Branchipolynoe symmytilida.

Polychaete species belonging to the genus Branchipolynoe are commensal with mussels from deep-sea hydrothermal vents and cold-seeps. Possessing hemoglobins (Hbs), the species B. symmytilida, which is found in the mussel Bathymodiolus thermophilus on the East Pacific Rise, is exceptional in a family normally devoid of respiratory pigments. In a previous paper we described two major coelomic extracellular hemoglobins with unique quaternary structures. Aiming to discern respiratory adaptations to the highly variable hydrothermal environment, this paper characterizes the functional properties of these Hbs and the coelomic fluid. The two major hemoglobins (C1 and C2) exhibit spectrophotometric characteristics of both intra- and extracellular hemoglobins. However, their amino acid content is very different from other known hemoglobins and is characterized by a high proportion of alanine and glycine (up to 40% cumulated in C1). C1 and C2 differ markedly by their cysteine content (0.8% and 13% respectively). The coelomic fluid exhibits a strong buffer capacity due to the high hemoglobin content (3 mM heme). In vitro, CO2 accumulation (up to 10-12 mM CO2 for PCO2 = 7.5 Torr) occurs with limited pH changes and is only partly accounted for by carbamino-Hb formation. The two hemoglobins exhibit high oxygen-affinities (P50 0.4 Torr for C1 and 0.9 Torr for C2, at 10 degrees C, pH 8) and a normal Bohr effect (phi values ranging from -0.54 and -0.37 at 10 degrees C, to -0.24 and -0.28 at 30 degrees C, for C1 and C2, respectively). Cooperativity values range from 0.8 to 1.9 for C1 and from 0.8 to 1.7 for C2. The temperature sensitivity of O2 affinity reflect deltaH values that decrease from -30 to -60 kJ x mol(-1) with increasing pH. C2 exhibits a slight specific effect of CO2 on oxygenation properties.

Oxygen binding and its allosteric control in hemoglobin of the pulmonate snail, Biomphalaria glabrata.

Pulmonate snails that experience extreme variations in gas tensions and temperatures possess extracellular, high-molecular mass ( approximately 1.7 x 10(6) Da) hemoglobins (Hbs) that are little known as regards oxygenation and allosteric characteristics. Biomphalaria glabrata hemolymph exhibits a high O2 affinity (half-saturation O2 tension = 6.1 mmHg; pH 7.7, 25 degreesC), pronounced Bohr effect (Bohr factor = -0.5), and pH-dependent cooperativity (Hill's cooperativity coefficient at half-saturation = 1.1-2.0). Divalent cations increase O2 affinity, Ca2+ exerting greater effect than Mg2+. Analyses in terms of the Monod-Wyman-Changeux model indicate novel O2 affinity control mechanisms. In contrast to vertebrate Hb, where organic phosphates and protons lower affinity via decreased O2 association equilibrium constant of Hb in low-affinity state (KT), and to extracellular annelid Hbs, where protons and cations primarily modulate O2 association equilibrium constant of Hb in high-affinity state (KR), in B. glabrata Hb, the Bohr effect is mediated predominantly via KR and the cation effect via KT, reflecting preferential, oxygenation-linked proton binding to oxygenated Hb and cation binding to deoxygenated Hb. CO2 has no specific (pH independent) effect. Nonlinear van't Hoff plots show temperature dependence of the overall heats of oxygenation, indicating oxy-deoxy heat capacity differences. The findings are related to possible physiological significance in pond habitats.

Interaction between haemoglobin and synthetic peptides of the N-terminal cytoplasmic fragment of trout band 3 (AE1) protein.

Two acidic peptides corresponding to the first 10 and 20 amino acid residues of the N-terminal, cytoplasmic fragment of rainbow trout band 3 (AE1) protein were synthesised in order to study their interaction with trout and human haemoglobin (Hb). The peptides did not influence the oxygen affinity of the main anodic trout Hb component (Hb IV) when tested at surplus peptide concentration ([peptide]/[Hb4]=16), at high and low ionic strength and at pH values ranging from 6.5 to 7.6. With human Hb, however, the 20-mer peptide markedly decreased the oxygen affinity and increased the Bohr effect. These data suggest that the trout band 3 peptide binds preferentially to the deoxy (T) conformation of human Hb, probably at the organic phosphate binding site in the central cavity between the beta-chains, which is known to be the binding site for the acidic N terminus of human band 3. In trout Hb IV, the presence of negatively charged Asp at position NA2 of the beta-chains (in contrast to positive or neutral residues in mammalian Hb) may weaken any interaction with the highly negatively charged peptides.

Hydrogen ion titrations of the anodic and cathodic haemoglobin components of the European eel Anguilla anguilla.

H+ titrations were conducted on the separated haemoglobin components of eel Anguilla anguilla in both the oxygenated and deoxygenated states. In anodic haemoglobin, the addition of GTP, and to a lesser extent C1-, increased the magnitude of the Haldane effect and shifted its maximum value into the in vivo pH range. Of the 22 histidine residues in the anodic component, only approximately seven were titratable, presumably the beta-chain residues at positions 41, 97, 109 and 146 (helical positions C7, FG4, G11 and HC3, respectively). In cathodic haemoglobin, a small negative Haldane effect was observed at pH values between 6.8 and 8.5 which disappeared in the presence of GTP (molar ratio 3:1 GTP:haemoglobin tetramer). GTP had virtually no effect on the buffer value at fixed oxygenation status, and the lowest buffer value was observed at in vivo pH values. No titratable histidine residues were observed in the cathodic component, indicating that all 14 histidines in this component are buried. We conclude that the anodic component, which constitutes two-thirds of the haemoglobin in the eel, plays the predominant role in CO2 transport and pH homeostasis in vivo.