Genetic diversity of coastal bottlenose dolphins revealed by structurally and functionally diverse hemoglobins

Studies of structure-function relationships in the respiratory proteins of marine mammals revealed unexpected variations in the number and types of hemoglobins (Hbs) present in coastal bottlenose dolphins, Tursiops truncatus. We obtained blood samples from free-ranging coastal bottlenose dolphins as a component of capture-release studies. We found that the oxygen-binding functions of bottlenose dolphin blood are poised between effector-saturated and unsaturated levels, enabling exercise-dependent shifts in oxygen transfer functions. Isolated bottlenose dolphin Hbs showed elevated pH sensitivities (Bohr effects) and appreciably lower oxygen affinities than adult human Hb in the absence of allosteric effectors. These properties may be an adaptive modification that enhances oxygen delivery during diving episodes when oxygen tensions and effector levels are low. The Hbs of individual dolphins showed similar oxygen affinities, responses to effectors, and expression of heme-heme interaction in oxygen binding, but differed in their redox potentials and rates of autoxidation. The heterogeneity suggested by these functional variations in Hbs of individual dolphins was born out by variations in the molecular weights and numbers of their alpha and beta globin chains. Although coastal bottlenose dolphins were expected to have a single type of Hb, the mass differences observed revealed considerable genetic diversity. There were multiple Hb forms in some individuals and differences in Hb patterns among individuals within the same community.     

Molecular Cloning and Evolutionary Analysis of Hemoglobin α-Chain Genes in Bats

Bats are the only mammals with the capacity for powered flight. When flying, they need abundant energy and oxygen. According to previous works, the hemoglobin (Hb) oxygen loading function of bats is insensitive to variations in body temperature, although different bat species have different heat sensitivity. We cloned Hb α-chain sequences from eight bat species to investigate whether they have different characteristics. We found that Hb in the bat lineages is under purifying selection, which accords with the importance of its function in bats. Three turn regions in bat Hb, however, have distinct evolutionary rates compared with those of other mammals, and the codons in these regions have an accelerated rate of evolution. These codons are under divergent selection in bats. These changes in Hb may have occurred in response to the physiological requirements of the species concerned, as adaptations to different lifestyles.     

The ratio of amount of haemoglobin to total surface area of erythrocytes in mammals

The literature provides all the data needed to calculate the ratio between the amount of haemoglobin and the total surface area of erythrocytes in 54 species of mammals ranging in body mass from 2.5 g to more than 1000 kg. Analysis shows that the concentration of haemoglobin (Hb; g%) does not depend on the body mass of the mammals studied. The number of erythrocytes in 1 mm³ of blood (RBC; 10⁶×mm⁻³) is significantly lower, and the diameter of these cells significantly higher, among larger mammals as opposed to smaller ones. The result is that the total surface area of erythrocytes in 1 mm³ of blood (TSAE; mm²×mm⁻³) is significantly lower among larger mammals, while the Hb/TSAE ratio (pg×μm⁻²) is significantly greater. These results point to the smaller size of erythrocytes of smaller mammals permitting much greater numbers to exist, thereby producing a greater TSAE and smaller Hb/TSAE ratio. The greater total surface area of red blood cells per unit volume of blood in small mammals can in turn be presumed to allow for full saturation of haemoglobin by oxygen, even where the period of contact between erythrocytes and air in the lungs is shorter than in their larger counterparts.

     

A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(β/δ)(1) and α(1)(β/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.     

Human hemoglobin Portland II (zeta 2 beta 2). Isolation and characterization of Portland hemoglobin components and their constituent globin chains

Two types of embryonic hemoglobins (Hb) containing zeta chains have been identified in the blood of several neonates of Chinese origin with homozygous alpha-thalassemia. In addition to Hb Portland I (zeta 2 gamma 2) which was previously reported, another embryonic hemoglobin has been detected and found to contain zeta chains and beta chains. It is being designated Hb Portland II and has the formula (zeta 2 beta 2). It has a mobility slightly slower than that of Hb A on starch gel electrophoresis at pH 8.6 and has been found in the hemolysates of blood of some but not all hydropic infants. Another component with a mobility faster than that of Hb A2 on starch gel has been isolated from the blood of some hydropic neonates. This latter component is postulated to be zeta 2 delta 2. The occurrence of Hb Portland I and Hb Portland II in these hydropic neonates is consistent with the hypothesis that, in the absence of normal alpha chain production, zeta chains are continued to be produced at later states of development than normal and form tetramers with each of the beta-like globin chains. Because Hb Portland II has not been found in blood from all hydropic neonates, we postulate that the presence of this hemoglobin in these fetuses may be correlated with the gestational age of the fetus at the time of birth.     

Molecular and physicochemical characterization of hemoglobin from the high-altitude Taiwanese brown-toothed shrew (Episoriculus fumidus)

Red-toothed shrews (subfamily Soricinae) exhibit the highest mass-specific rates of O? consumption recorded among eutherian mammals, though surprisingly no data appears to be available on the functional characteristics of their hemoglobin (Hb). As a first step in addressing this shortcoming, we investigated the O? binding characteristics of Taiwanese brown-toothed shrew (Episoriculus fumidus) Hb and its temperature and pH dependence in the absence and presence of anionic red blood cell effectors. Although comparative data regarding the intrinsic O? affinity of other shrew species are currently unavailable, our data suggest that the sensitivity of this high-elevation endemic species' Hb to allosteric effector molecules is similar to that of the two lowland species of white-toothed (crocidurine) shrews examined to date. The efficient exploitation of blood O? reserves by E. fumidus appears to be achieved via synergistic modulation of O? affinity by Cl? and organic phosphates that moreover dramatically lowers the overall enthalpy of oxygenation of their Hb. Oxygen unloading is presumably further enhanced by a relatively high Bohr effect (ΔLog P??/ΔpH = -0.69) and marked reduction in the titratable histidine content (predicted low proton buffering value) of the component globin chains relative to human HbA. Notably, however, the limited data available suggest these latter attributes may be widespread among shrews and hence likely are not adaptations to chronic altitudinal hypoxia per se.     

Generation of species-specific antihemoglobin antibodies by immunization with synthetic peptides of human hemoglobin

Four peptides (7–16 residues) representing nonconserved regions of human hemoglobin (Hb) were selected for synthesis by comparison of the amino acid sequence of human Hb with those of the most common domesticated animals. Mouse antisera resulting from immunization with the synthetic peptides were investigated for binding to a panel of animal Hbs using solid-phase radioimmunoassay (RIA). One of the peptides elicited antibodies which bound specifically to human Hb, but not to any Hb of the nonprimate animals tested. The results show that the peptide immunogen chosen on the basis of dissimilarity between regions of different species is useful for the generation of species-specific antibodies. Such antibodies could serve as valuable tools for clinical screening of fecal occult blood trait and for forensic identification of bloodstains of human origin.     

Features of photochemical properties of hemoglobin under native conditions

The photochemistry of human hemoglobin (Hb) in the blood and blood serum (lambda = 254-578 nm) was studied, using spectrophotometric methods. The Hb photochemistry is a complex set of photoreactions leading to successive photoconversions of Hb forms: from oxy- to met- to deoxy- and, finally, to carboxy-form. The photodestruction of Hb and the photoreactions involving other serum proteins were found to occur simultaneously. In the blood Hb photomodifications are localized directly in erythrocytes. The conditions necessary for the photo--induced rupture of erythrocyte membranes and the subsequent release of Hb into the blood plasma, were determined. Although the general characteristics of Hb photochemistry are the same for model systems and for native conditions, there are some distinctions in the effectiveness of the photoconversion. It seems likely that the observed effects are due to the antioxidant properties of the serum. These properties may be the cause of the inhibition of blood photohemolysis upon irradiation (lambda greater than or equal to 300 nm).     

Running, swimming and diving modifies neuroprotecting globins in the mammalian brain

The vulnerability of the human brain to injury following just a few minutes of oxygen deprivation with submergence contrasts markedly with diving mammals, such as Weddell seals (Leptonychotes weddellii), which can remain underwater for more than 90 min while exhibiting no neurological or behavioural impairment. This response occurs despite exposure to blood oxygen levels concomitant with human unconsciousness. To determine whether such aquatic lifestyles result in unique adaptations for avoiding ischaemic-hypoxic neural damage, we measured the presence of circulating (haemoglobin) and resident (neuroglobin and cytoglobin) oxygen-carrying globins in the cerebral cortex of 16 mammalian species considered terrestrial, swimming or diving specialists. Here we report a striking difference in globin levels depending on activity lifestyle. A nearly 9.5-fold range in haemoglobin concentration (0.17-1.62 g Hb 100 g brain wet wt(-1)) occurred between terrestrial and deep-diving mammals; a threefold range in resident globins was evident between terrestrial and swimming specialists. Together, these two globin groups provide complementary mechanisms for facilitating oxygen transfer into neural tissues and the potential for protection against reactive oxygen and nitrogen groups. This enables marine mammals to maintain sensory and locomotor neural functions during prolonged submergence, and suggests new avenues for averting oxygen-mediated neural injury in the mammalian brain.     

A recombinant polymeric hemoglobin with conformational,functional, and physiological characteristics of an in vivo O2 transporter

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.     

The kinetics of the diffusion-controlled reaction of the binding of carbon monoxide to hemoglobin in glycerol-water mixtures of high viscosity

The kinetics of the binding of carbon monoxide to human hemoglobin and to ferrous horseradish peroxidase (HRP) have been studied by flash photolysis in mixtures of glycerol and water over a wide range of temperature and solvent viscosities. This was done in order that the influence of diffusion-control on the association rates could be determined. The binding of CO to HRP which is much slower than binding to Hb was devoid of diffusion effects. By contrast, the fast and slow phases of binding to Hb in the high viscosity solvents both displayed curved Arrhenius plots, consistent with a change from a chemical activationcontrolled process in the high temperature region to a diffusion-controlled process in the low temperature region. Analyses of the curved Arrhenius plots indicated that in the low temperature diffusion-controlled region, the activation enthalpy is similar to the activation energy of viscosity of the solvent, as might be expected for a diffusion-controlled reaction.Curve fitting of rate-temperature-viscosity data, assuming simultaneous chemical activation and diffusion-control, yielded factors by which the diffusion rate constants differ from that for reaction between uniformly reactive spheres of equal radii. For the fast Hb reaction, observed upon partial photolysis, this factor varies from 0.02 to 1.1, depending upon the solvent composition. For the slow Hb reaction, observed upon higher degrees of photolysis, this factor was 0.03 and 0.04. These factors were rationalized in terms of fractional surface reactivities and of a maximum allowable solid angle of entry of reactant to the binding site. It was concluded that the steric hindrance of T-state Hb (slow reaction) is much greater than R-state Hb (fast reaction).     

Acetylation of human fetal hemoglobin occurs throughout erythroid cell maturation

The biosynthesis of human acetylated fetal hemoglobin (Hb F1) has been examined by incubating the following cell types with [3H]leucine: (a) burst-forming unit erythroid cells cultured from umbilical cord mononuclear cells, (b) infant bone marrow, (c) umbilical cord blood, and (d) peripheral blood cells from adults with elevated fetal hemoglobin. Newly synthesized Hb F1 was 18-20% that of Hb F0 in burst-forming unit erythroid cells which were immature, mature, or in an intermediate state of development. In infant marrow and in infant and adult peripheral blood the extant Hb F1 comprised 10.8 +/- 1.8% of the total Hb F. In marrow cells the specific radioactivity (cpm/mg) of Hb F1 was 1.4-2.0-times greater than that of Hb F0. In peripheral blood cells these ratios were slightly greater. [3H]Leucine-labeled infant bone marrow, umbilical cord blood, and adult peripheral blood cells were subjected to density gradient ultracentrifugation. The ratios of specific radioactivity for Hb F1/Hb F0 increased from 1.0-1.8 in the lightest cell zone to 5.2-9.0 in the more dense cells. Thus the biosynthesis of Hb F1 is enhanced in cells which are more mature than those responsible for the bulk of hemoglobin synthesis, and the acetylation of Hb F provides a marker of erythroid cell maturation.     

Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer Dissociations

Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer → dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer → monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or re-association reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development.     

Respiratory Adaptations in Marine Mammals

This paper is a discussion of some of the possible structuraland functional modifications of the lung which represent adaptationsin mammals living in the sea. Lung capacities of marine mammalsseem to be larger than terrestrial mammals especially if theyare compared on a lean weight basis. It is proposed that atleast in some this represents an important increase in buoyancywhich enables these mammals to rest at sea. The importance ofthe lung as an O₂ store during dives is considered, and it seemsthat it would be important only to those species that have alow breath-hold tolerance. In their case the O₂ present in thefully inflated lung is from four times to equal that in theblood. In those species with a large breath-hold tolerance thelung O₂ store is a small fraction of blood stores. Several experimentsare discussed which indicate that during dives to depth gasexchange between the blood and lungs is low. One of the reasonssuggested is compression collapse of the alveoli. This occursbecause of the apparent rigidity of the airways which even inthe terminal segments possess an unusual amount of muscularor cartilaginous support. The reinforcement insures that duringcompression the airways will not occlude and trap gas in thealveoli. In fact, in some species, especially otariids and cetaceans,the airways seem overly strong and an additional function issuggested. Studies of mechanical properties of sea lion andwhale lungs show that they may be capable of high expiratoryflow rates at low volumes. This feature of the lung would makepossible an exchange of a large gas volume in very short periods.Such an ability is consistent with the ventilatory behaviorof many marine mammals.     

Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System

All vertebrates except cold-water ice fish transport oxygenvia hemoglobin packaged in red blood cells (RBCs). VertebrateRBCs vary in size by thirtyfold. Differences in RBC size havebeen known for over a century, but the functional significanceof RBC size remains unknown. One hypothesis is that large RBCsare a primitive character. Agnathans have larger RBCs than domammals. However, the largest RBCs are found in urodele amphibianswhich is inconsistent with the hypothesis that large RBCs areprimitive. Another possibility is that small RBCs increase bloodoxygen transport capacity. Blood hemoglobin concentration ([Hb])and mean RBC hemoglobin concentration (MCHC) increase from Agnathato birds and mammals. However, the changes in [Hb] and MCHCdo not parallel changes in RBC size. In addition, RBC size doesnot affect blood viscosity. Thus, there is no clear link betweenRBC size and oxygen transport capacity. We hypothesize thatRBC size attends changes in capillary diameter. This hypothesisis based on the following observations. First, RBC width averages25% larger than capillary diameter which insures cell deformationduring capillary flow. Functionally, RBC deformation minimizesdiffusion limitations to gas exchange. Second, smaller capillariesare associated with increased potential for diffusive gas exchange.However, smaller capillaries result in higher resistances toblood flow which requires higher blood pressures. We proposethat the large capillary diameters and large RBCs in urodelesreflect the evolutionary development of a pulmonary vascularsupply. The large capillaries reduced systemic vascular resistancesenabling a single ventricular heart to supply blood to two vascularcircuits, systemic and pulmonary, without developing high pressureson the pulmonary side. The large RBCs preserved diffusive gasexchange efficiency in the large capillaries.     

Side-necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA-derived primary structures and X-ray crystal structures of Hb A

Red blood cells of yellow-spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical α(A) -globin polypeptides and two identical β-globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P4?2?2), produced under neutral pH (pH 7-8) conditions; and form B, hexagonal crystals (space group, P6?22), produced under high alkaline pH (pH 11-13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X-ray crystallographic diffraction at a resolution of 2.20 ? for form A and 2.35 ? for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 ?. These low values indicate the crystal structures resemble each other. Our data on X-ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.     

Adaptation to Hypoxia by Increased HbO<Subscript>2</Subscript> Affinity and Decreased Red Cell ATP Concentration

THERE is a decrease in the O₂ affinity of mammalian haemoglobin (Hb) as the levels of 2,3-DPG or ATP are increased, which is explained by an allosteric effect on the HbO₂ binding^1,2. Similar observations on amphibians³ and fish⁴, which have molar ratios of ATP to Hb similar to those of DPG to Hb in mammals, suggest that red cell organic phosphates modulate Hb function in all vertebrates. The adaptation of mammals to various hypoxic stresses involves reduced HbO₂ affinity^5–9, the attendant increase in O₂ “unloading” capacity being mediated by an increase in the concentration of red cell 2,3-DPG. We have found the opposite response in hypoxic fish and suggest that an increased O₂ affinity results in increased O₂ transport for the fish.     

Spectroscopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera:Notonectidae).

Hemoglobin (Hb) isolated from the backswimmer Buenoa margaritacea has been analyzed spectroscopically. The met form at pH less than 6 shows a 30nm red shift in the Qv and Qo bands and a 5nm red shift in the Soret band compared to mammalian Hb, while only minor differences are seen in the spectra of the CO and O2 adducts of Hb from Buenoa and mammals. EPR spectra of the metHb show a superposition of signals; at low pH they are mainly of axial high-spin character, while at high pH a low-spin signal predominates with an O-type g-tensor (2.54, 2.61, 1.85) comparable to that of hydroxy myoglobin. Infrared spectra of Hb12C-16O at pH 8.2 reveal two major absorption bands at 1934 cm-1 and 1967 cm-1, which shift to 1892 cm-1 and 1923 cm-1, respectively, for Hb12C-18O. As isolated the Buenoa Hb consists of several isozymes, all of which have a histidine as the proximal ligand of the heme iron.     

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50-250 microg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body's endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.