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.     

Hemoglobin function in the vertebrates: An evolutionary model

Comparative data on quaternary structure, cooperativity, Bohr effect and regulation by organic phosphates are reviewed for vertebrate hemoglobins. A phylogeny of hemoglobin function in the vertebrates is deduced. It is proposed that from the monomeric hemoglobin of the common ancestor of vertebrates, a deoxy dimer, as seen in the lamprey, could have originated with a single amino acid substitution. The deoxy dimer has a Bohr effect, cooperativity and a reduced oxygen affinity compared to the monomer. One, or two, additional amino acid substitutions could have resulted in the origin of a tetrameric deoxy hemoglobin which dissociated to dimers on oxygenation. Gene duplication, giving incipient alpha and beta genes, probably preceded the origin of a tetrameric oxyhemoglobin. The origin of an organic phosphate binding site on the tetrameric hemoglobin of an early fish required only one, or two, amino acid substitutions. ATP was the first organic phosphate regulator of hemoglobin function. The binding of ATP by hemoglobin may have caused the original elevation in the concentration of ATP in the red blood cells by relieving end product inhibition of ATP synthesis. The switch from regulation of hemoglobin function by ATP to regulation by DPG may have been a consequence of the curtailment of oxidative phosphorylation in the red blood cell. The basic mechanisms by which ATP and DPG concentrations can respond to strss on the oxygen transport system were present before the origin of an organic phosphate binding site on hemoglobin. A switch from ATP regulation to IP5 regulation occurred in the common ancestor of birds.     

A membrane-bound hemoglobin from gills of the green shore crab Carcinus maenas

Most hemoglobins serve for the transport or storage of O(2). Although hemoglobins are widespread in "entomostracan" Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe(2+) and an oxygen affinity of P(50) = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O(2) supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.     

The binding of hemoglobin to membranes of normal and sickle erythrocytes

The binding of hemoglobins A, S, and A₂ to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with ¹⁴C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A₂, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4–22 °C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied.An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites.These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.     

Hemoglobins, XXVIII. Phosphate-protein-interaction, gene expression and function: the genetic and allosteric control of the oxygen affinity of the fetal blood (author's transl)

This work describes possible molecular mechanisms concerning the control of oxygen affinity in fetal blood of mammalia. There is a genetic control of oxygen affinity through a fetal gene: at constant phosphate concentration (Hb less than P2-glycerate) in humans there is a hemoglobin with only five binding sites to 2,3-bisphosphoglycerate, resulting in an increased oxygen affinity. In several species (sheep, cattle, goat) with Met-Leu as the N-terminal group of the beta-chains, the 2,3-bisphosphoglycerate binding sites are deleted in positions beta 1 and beta 2, so that the regulation is phosphate-independent and thus providing a fetal hemoglobin with an increased oxygen affinity. The allosteric control is observed in pigs. In the postembryonal development "adult" hemoglobin with seven contacts (beta-chains) is demonstrated. The increased oxygen affinity is achieved here by a reduced biosynthesis of 2,3-bisphosphoglycerate (Hb greater than P2-glycerate) (Rapoport-Luebering-cycle). The functional control is discussed with respect to the ontogeny of the hemoglobins.     

Recombinant hemoglobins with low oxygen affinity and high cooperativity

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in hemoglobin.     

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Studies on the interaction of zinc with human hemoglobin

Zn has previously been shown to increase the oxygen affinity of both normal and sickle red blood cells. Experiments are presented which demonstrate that the oxygen affinity effect of Zn is due to a Zn-hemoglobin binding mechanism rather than a Zn-2,3 diphosphoglycerate binding mechanism. Further a large shift (6 mm Hg) in the oxygen affinity of a red cell-saline suspension occurs with a low Zn/hemoglobin (tetramer) molar ratio (0.4). Zn had no influence on the Bohr effect of hemoglobin but it did decrease the Hill coefficient. Hemoglobin binding experiments using partially purified hemoglobin indicated that Zn can bind to more than one amino acid residue but it appears that the amino acid residue with the highest binding capacity for Zn is also the residue involved in the oxygen affinity effect of Zn. Hydrogen ion concentration (pH 5–8) had no influence on the Zn/hemoglobin ratios obtained in these binding experiments. The possible (and the improbable) Zn binding sites on the hemoglobin molecule are discussed.     

A fetal-maternal shift in the oxygen equilibrium of hemoglobin from the viviparous caecilian,Typhlonectes compressicauda

• 1. The equilibria and kinetics of oxygen binding by blood and hemoglobin from adult and fetal caecilians,Typhlonectes compressicauda, have been measured.
• 2. The oxygen affinity of fetal blood is higher than that of adult blood.
• 3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each.
• 4. Adult and fetal hemoglobins have identical oxygen equilibria. Stripped hemoglobins have a high oxygen affinity and no Bohr effect between pH 6.5 and 10.0. An “acid”, reversed Bohr effect is present below pH 6.5. The addition of 1 mM ATP reduces the oxygen affinity markedly and produces a moderate, normal Bohr effect.
• 5. The major nucleoside triphosphate in fetal and adult erythrocytes is adenosine triphosphate: about 10% of the nucleoside triphosphates is guanosine triphosphate. Adult erythrocytes contain 3 times as much ATP as do the fetal erythrocytes.
• 6. The fetal to maternal shift in the oxygen equilibrium is mediated entirely by the difference in ATP content of the maternal and fetal red blood cells.

     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

Genetic variation and functional properties of Atlantic cod hemoglobins: introducing a modified tonometric method for studying fragile hemoglobins

Hemoglobin polymorphism in Atlantic cod has been investigated with respect to physiological performance at 10, 15 and 20 degrees C applying a modified tonometric method for O2 equilibrium analysis with full control of the equilibrating gas mixture. The results did not indicate any dissociation of the hemoglobins by a reduction in cooperativity and a parallel increase in affinity during the analytical procedure in contrast to the original tonometric method. With the applied preparation technique, we could store the hemolysate for 70 days at -25 degrees C without any significant changes in the O2 binding properties (P < 0.05) demonstrating the high quality of this procedure for analysing fragile fish hemoglobins. The present investigation demonstrates that the oxygen affinity of the hemoglobins varied between the genotypes. At all temperatures, except 20 degrees C and pH 8.0, Hb-I(2/2) had a higher O2 affinity than Hb-I(1/1). These results conform with previous results (16), suggesting Hb-I(2/2), the genotype which is the dominant allele in northern areas, to be the most efficient O2 carrier at low temperatures. The highest O2 affinity, however, was found for Hb-I(2/2b), supporting the results of Fyhn et al. (9), that this genotype is more restricted to coastal and warmer water and thus a better marker of the coastal population. Our results further suggest a correlation between genotype specific growth rates and oxygen affinities at all temperatures studied, with the highest growth rates observed in those genotypes having the highest O2 affinities. In conclusion, the hemoglobin polymorphism of cod seems to be correlated with physiological performance.     

Direct measurement of equilibrium constants for high-affinity hemoglobins

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.     

Interactions of nicotinamide adenine dinucleotides with varied states and forms of hemoglobin

Spectrofluorometric techniques were used to quantify NADPH-hemoglobin interactions based on the quenching of NADPH fluorescence upon binding to hemoglobin. Fluorometric titrations were carried out with hemoglobin in varied states and with hemoglobins in which the beta-chain anion site is altered. At pH 6.5 in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, NADPH binds with high affinity, Kd = 1.03 microM, to deoxy human hemoglobin tetramers. Lower affinity binding of NADPH occurs as the beta-chain anion-binding site is discharged by increasing the pH. Moreover, the cofactor binds in a 1:1 ratio to deoxy tetramers, inositol hexaphosphate binds competitively, and binding is decreased in hemoglobins whose structural alterations result in decreased effects of 2,3-diphosphoglycerate. The cofactor binds to oxidized (met) hemoglobin with an estimated Kd of 33.3 microM but has little or no affinity for the oxy form. These results indicate that NADPH binds at the beta-chain anion-binding site and can be considered as a fluorescent analog of 2,3-diphosphoglycerate. Fluorescence measurements gave no indication of NADPH binding to deoxygenated ferrous or ferric myoglobin. Reductive processes within the erythrocyte, such as reduction of met hemoglobin and hemoglobin-catalyzed enzymatic reactions, may be affected by the significant binding of the reduced cofactor to both deoxygenated and oxidized hemoglobin. Cofactor-hemoglobin interactions predict a shift in redox potential as red cells become oxygenated, which may account for unexplained oxygen-linked shifts in red cell metabolism.     

Allosteric effect of water in fish and human hemoglobins

Prompted by the reported lack of solvation effects on the oxygen affinity of fish (trout I) hemoglobin that questioned allosteric water binding in human hemoglobin A (Bellelli, A., Brancaccio, A., and Brunori, M. (1993) J. Biol. Chem. 268, 4742-4744), we have investigated solvation effects in fish and human hemoglobins by means of the osmotic stress method and allosteric analysis. In contrast to the earlier report, we demonstrate that water potential does affect oxygen affinity of trout hemoglobin I in the presence of inert solutes like betaine. Moreover, we show that upon oxygenation electrophoretically anodic hemoglobin from trout and eel bind a similar number of water molecules as does human hemoglobin A, whereas the cathodic hemoglobins of trout and eel bind smaller, but mutually similar, numbers of water molecules. Addition of cofactors strongly increases the number of water molecules bound to eel hemoglobin A (as in human hemoglobin) but only weakly affects water binding to eel hemoglobin C.     

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.