The hemoglobin system of the serpent eel Ophisurus serpens: structural and functional characterization

The hemoglobin system of the serpent eel Ophisurus serpens was structurally and functionally characterized with the aim of comparing it to the hemoglobin system of other fish species, as oxygen loading under the severe habitat conditions experienced by O. serpens could have necessitated specific adaptation mechanisms during evolution. The hemoglobin system of O. serpens includes one cathodic and four anodic components. The molecular mass of the α and β chains of the cathodic component as well as the 2 α and 4 β of the anodic components were determined. Analysis of the intact α and β chains from cathodic hemoglobin and their proteolytic digestion products by high-resolution MS and MS/MS experiments resulted in 92 and 95 % sequence coverage of the α and β globins, respectively. The oxygen binding properties of both hemoglobin components were analyzed with respect to their interactions with their physiological effectors. Stripped cathodic hemoglobin displayed the highest oxygen affinity among Anguilliformes with no significant effect of pH on O₂-affinity. In the presence of both chloride and organic phosphates, O₂-affinity was strongly reduced, and cooperativity was enhanced; moreover, cathodic hemoglobin contains two indistinguishable GTP-binding sites. Stripped anodic hemoglobins exhibited both low O₂-affinity and low cooperativity and a larger Bohr effect than cathodic hemoglobin. The cathodic hemoglobin of O. serpens and the corresponding component of Conger conger share the greatest structural and functional similarity among hemoglobin systems of Anguilliformes studied to date, consistent with their phylogenetic relationship.     

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.     

Characterization of the hemoglobins of the adult brushtailed possum, Trichosurus vulpecula (Kerr) reveals non-genetic heterogeneity

The hemoglobins contained within the red blood cells of the adult brushtail possum exhibited cooperative (n=2.6) oxygen binding curves with an associated p50 of 38 mm Hg at pH 7.4 and a large Bohr effect (-0.60). Stripped hemolysate showed a Bohr effect of -0.27, and was sensitive to added DPG (K=56 micromol L(-1)), ATP (K=130 micromol L(-1)), and chloride ions. Four isoforms of hemoglobin were identified using isoelectric focusing. Mass spectrometry indicated that all four isoforms most likely represent the same gene products which have differentially undergone post-translational deamidation and glutathionylation. The oxygen binding characteristics of three isolated isohemoglobins have been determined.     

Functional properties of the hemoglobin from the South American snake Mastigodryas bifossatus

The hemolysate of Mastigodryas bifossatus shows two major hemoglobins with very close isoelectric points, and four different globin chains. The stripped hemolysate exhibits a low alkaline Bohr effect (Δlog P₅₀/ΔpH = −0.30 between pH7 and 8) and a decrease of the co-operativity from 2.3 to unity when the pH increases from 6.15 to 8.5. In the presence of ATP, large changes in the oxygen affinity and co-operativity are observed. The Bohr effect rises to −0.46 and the n₅₀ values stay at around 3 in the pH range 6–9. An increase in temperature induces a large decrease in the oxygen affinity for the stripped hemolysate. In the pH range between 7.5 and 8.5, the values of AH in kcal/M are around 10 fold larger for the stripped protein than for the protein in the presence of ATP. Measurements of rapid kinetics of oxygen dissociation and carbon monoxide binding reflect the ATP sensitivity observed in equilibrium experiments.     

Some functional and structural properties of Bufus paracnemis and Pipa pipae hemoglobins

1. Hemoglobin from terrestrial and an aquatic amphibia Bufo paracnemis and Pipa pipae respectively both living in the same region (Belém, Pará) were compared. 2. The number of hemolysate components were determined by starch-gel electrophoresis and CMC-chromatography. P. pipae hemoglobin presented 4 components and B. paracnemis 2, all of anodic mobility. 3. Functional properties of the hemoglobins (oxygen affinity. Bohr effect, carbon monoxide equilibrium) were determined and compared. 4. The differences found in the functional properties were correlated with the different habitat: aquatic or terrestrial for the amphibia. 5. The study of the oxygen functional properties of the hemoglobin showed in the stripped proteins of P. pipae in the presence of ATP, an oxygen affinity (P50 = 7.24 mmHg) that of B. paracnemis at the same pH (P50 = 15.84 mmHg). At high pH the P50 values are different being 15.84 mmHg for the terrestrial frog and 5 mmHg for P. pipae haemoglobin both at pH 8. In addition Bohr effect was noted only in P. pipae hemolysate in the presence of ATP. 6. The CO-equilibrium affinity constant in the presence of ATP are similar in both frogs, of about, log C50 = -6.9. The ratio Pco/Po2 for B. paracnemis hemoglobin was about 300 whereas for that of P. pipae was about 100 only. 7. The kinetic study of reactive sulfhydryl groups in both frog hemoglobin with 4-PDS (specific SH group reagent was used and shown high pseudo-first order constant for B. paracnemis hemoglobin) (k' = 0.46/min) either in presence or not of ATP, compared for that of P. pipae where values were K' = 0.003/min in the stripped protein and k' = 0.014/min in the presence of ATP. 8. Denaturation kinetic studies of the hemoglobin with sodium benzoate was performed and the results were compared with that of Rana catesbeiana, i.e. the pseudo-first order of the hemoglobin denaturation reaction are low for the aquatic P. pipae adult although for the B. paracnemis high molecular resistant was also noted. For P. pipae hemoglobin, nevertheless with ATP such resistance becomes higher. This does not occur with B. paracnemis hemoglobins.     

Functional characterization of the single hemoglobin of the migratory bird Ciconia ciconia

Hemolysate from white stork displayed a single hemoglobin component, thus resulting into two bands and two globin peaks in dissociating PAGE and reversed phase-HPLC, respectively. Stripped hemoglobin showed an oxygen affinity higher than that of human HbA, a small Bohr effect, and a cooperative oxygen binding. A small decrease of oxygen affinity, of the same extent in all the pH range examined, was observed by addition of chloride, thus indicating an unusual chloride-independent Bohr effect (DeltalogP50/Deltalog pH=-0.24). Saturating amounts of inositol hexakisphosphate, largely decreased hemoglobin-oxygen affinity (DeltalogP(50)=1.17 at pH 7.0), and increased the extent of its Bohr effect (DeltalogP50/DeltalogpH=-0.45). The phosphate binding curve allowed to measure a very high overall binding constant (K=1.18 x 10(5) M(-1)). The effect of temperature on the oxygen affinity was measured, and the enthalpy change of oxygenation resulted almost independent on pH. Structural-functional relationships are discussed by considering some amino acid residues situated at alpha1/beta1 and alpha1/beta2 interfaces, such as alpha38 and alpha89 positions. The presence of only one hemoglobin component, a rare event among birds, and its functional properties have been related to the physiological oxygen requirements of this soaring migrant bird and to its technique of flight during migration.     

Multiple hemoglobins of the cutthroat trout, Salmo clarki

Nine hemoglobins were purified from blood of Salmo clarki by ion-exchange chromatography and preparative isoelectric focusing. The subunit structures of eight of the purified hemoglobins were studied by electrophoresis of globins in the presence of urea. Six are alpha 2 beta 2 tetramers while two appear to be heterotetramers of the type alpha alpha' beta 2 and alpha alpha' beta beta'. The effects of pH, nucleotides, and temperature on the oxygen equilibria of the purified hemoglobins were studied. Five hemoglobins with isoelectric points from 9.1 to 7.1 and one minor hemoglobin with an isoelectric point of 5.9 appear to have essentially identical oxygen binding properties. All have similar oxygen equilibria which are independent of pH and temperature and not affected by saturating amounts of ATP. Another minor hemoglobin with an isoelectric point below 5.9 has similar oxygen equilibria except for a possible pH dependence. Two hemoglobins, with isoelectric points of 6.5 and 6.4, have oxygen binding properties which are strongly pH and temperature dependent. Addition of ATP or GTP causes a large decrease in the oxygen affinity without affecting the cooperativity of oxygen binding. The effect of GTP is slightly greater than that of ATP. No significant differences were observed in the oxygen equilibria of these two hemoglobins. The red blood cells of S. clarki were found to contain large amounts of both ATP and GTP, with an ATP:GTP ratio of 3:1. Both nucleotides may be important modulators of hemoglobin oxygen affinity in S. clarki, in contrast to the situation in S. gairdneri, in which red blood cell GTP concentrations are considerably lower. The presence of six or possibly seven hemoglobins with identical oxygen binding properties in S. clarki suggests that, to a large extent, the physiological role of multiple hemoglobins in this species involves phenomena not directly related to the oxygen binding properties of the hemoglobins.     

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.     

Structural and functional studies of the king salmon, Oncorhynchus tshawytscha, hemoglobins

Vertical starch-gel electrophoresis at pH 8.6 revealed extensive hemoglobin multiplicity with several distinct cathodal and anodal hemoglobin components. Anodal hemoglobin components are present throughout the life cycle of the king salmon. Additional cathodal components are found in the adult fish. Cathodal hemoglobin components exhibited a higher oxygen affinity (P50 = 10.2 mm at 13 degrees C, pH 7.3) than the anodal hemoglobin components (P50 = 21.8 mmHg at 13 degrees C). Oxygen binding of the anodal hemoglobins are sensitive to pH, temperature, organic phosphates (ATP and GTP), as well as, ionic strength; binding of oxygen to the cathodal hemoglobins is independent of pH and not affected by organic phosphates. Anodal hemoglobin components are less resistant to thermal denaturation over the pH 6.0 to 8.0 range. Isothermal urea denaturation of separated anodal and cathodal hemoglobin fractions of the king salmon indicate inherent differences in the stabilization energies of these hemoglobins. Autoxidation of these hemoglobins occurs around pH 7.0 and below, as well as, in the presence of increasing Cl- concentrations.     

Intraerythrocytic organic phosphates of fetal and adult seaperch (Embiotoca lateralis): Their role in maternal-fetal oxygen transport

Summary The viviparous seaperch,Embiotoca lateralis, has unique fetal and adult hemoglobins. Stripped fetal hemoglobin has a higher oxygen affinity than stripped adult hemoglobin at pH 6.5–7.1. The oxygen affinities of both adult and fetal hemoglobins are lowered allosterically by ATP at pH 7.1. Both fetal and adult seaperch erythrocytes include approximately 82% ATP and 18% GTP of the total nucleotide triphosphates (NTP) with a trace of AMP. No 2,3-diphosphoglycerate or inositol polyphosphate was detected. Mid- and late-gestation erythrocytes contain less NTP/mole hemoglobin tetramer than do adult cells. The effective NTP concentration in adult cells is higher than that of the fetal erythrocytes even when the intracellular concentration of Mg²⁺, which complexes with NTP, is accounted for. The difference in adult and fetal intraerythrocytic NTP concentration should enhance transfer of oxygen from maternal to fetal blood. Thus, the teleostEmbiotoca lateralis may employ a dual mechanism in maternal-fetal oxygen transfer. A difference in fetal and maternal hemoglobin structure and oxygen affinities is enhanced by a difference in their respective intraerythrocytic organic phosphate concentrations.     

Biochemical aspects of the adaptation of hemoglobin-oxygen affinity of eels to hypoxia.

The heterogeneity and oxygenational properties of eel hemolysates show no acclimatory response to hypoxia as is found in whole blood. Guanosine triphosphate (GTP) plays a greater role than ATP in acclimation to hypoxia, both by virtue of larger concentrational changes and its greater effect on oxygen affinities. The greater anion sensitivity of the cathodic compared to the anodic components appears to be of basic significance in the adaptation of hemoglobin oxygen affinity to environmental oxygenation.     

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.     

Subunit interactions of Glycera dibranchiata hemoglobin.

The coelomic cells of the polychaete annelid Glycera dibranchiata contain two hemoglobins. The monomer hemoglobin fraction is composed of one major component and two minor components as determined by starch gel electrophoresis and isoelectrofocusing, but is homogeneous as to subunit size as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymer hemoglobin fraction has and initial molecular weight of Mn = 125,000 as determined by osmometry, but exhibits an increased state of aggregation upon storage. The quaternary structure of the polymer is constituted of monomeric subunits in a non-covalent state of aggregation as demonstrated by its subunit dissociation inthe presence of propyl urea. The oxygen affinity of the polymer is lower than the monomer but increases with deaggregation. The Bohr effect is present only in the polymer. Cooperativity is also characteristic of the polymer and is pH-dependent. Interestingly, cooperativity increases with intermediate states of polymer deaggregation. By far, the main organic phosphate component of the coelomic red cells is ATP accompanied by small amounts of ADP and GTP. No modulating effect of ATP on the oxygen equilibrium of either polymer or total hemolysate was found.     

Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

• 1.1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components.
• 2.2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation.
• 3.3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect.
• 4.4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity.
• 5.5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

     

The effect of 4-isothiocyanatobenzene sulfonic acid on the oxygen affinity of human hemoglobin

Human hemoglobin reacts with 4-Isothiocyanatobenzene sulfonic acid at the four amino groups of the N-terminal valines. The modified protein shows a decreased oxygen affinity over a wide pH range, a reduced alkaline Bohr effect, decreased co-operativity, and a reduced effect of inositol hexasulfate on the oxygen affinity.     

Purification and characterization of the hemoglobin components of Adriatic sturgeon (Acipenser naccarii) blood

The oxygen binding properties of Acipenser naccarii hemoglobins have been investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes and hemolysate in the presence of the physiological cofactors GTP and chloride ions. In particular, the oxygen equilibrium reveal a very low sensitivity of the hemolysate to the chloride ions and temperature while in the presence of organic phosphates their oxygen affinity decreased strongly. The electrophoretic analysis of the hemolysate of the sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. The partial amino acid sequence of the a and P chain of the single hemoglobins was also established.     

Role of Bohr group salt bridges in cooperativity in hemoglobin.

Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity.     

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.     

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.     

Allosteric water and phosphate effects in Hoplosternum littorale hemoglobins.

This paper reports the results obtained using the osmotic stress method applied to the purified cathodic and anodic hemoglobins (Hbs) from the catfish Hoplosternum littorale, a species that displays facultative accessorial air oxygenation. We demonstrate that water potential affects the oxygen affinity of H. littorale Hbs in the presence of an inert solute (sucrose). Oxygen affinity increases when water activity increases, indicating that water molecules stabilize the high-affinity state of the Hb. This effect is the same as that observed in tetrameric vertebrate Hbs. We show that both anodic and cathodic Hbs show conformational substrates similar to other vertebrate Hbs. For both Hbs, addition of anionic effectors, especially chloride, strongly increases the number of water molecules bound, although anodic Hb did not exhibit sensitivity to saturating levels of ATP. Accordingly, for both Hbs, we propose that the deoxy conformations coexist in at least two anion-dependent allosteric states, T(o) and T(x), as occurs for human Hb. We found a single phosphate binding site for the cathodic Hb.