The primary structure of the hemoglobin of the electric eel (Electrophorus electricus)

The blood of the Electric Eel contains only one hemoglobin component. The primary structures of the alpha- and beta-chains are presented. These were separated by high-performance liquid chromatography, using a new kind of buffer system. The alpha-chains are acetylated, and consist of 142 residues, while the beta-chains are not blocked, and consist of 147 residues. The phylogenetic distances between these and the alpha- and beta-chains of human hemoglobin are 48 and 50% amino-acid exchanges, respectively. The relationship between primary structure and the Bohr effect and Root effect is discussed, especially the significance of the serine found in position F9 beta.     

The primary structure of the hemoglobin of the dogfish shark (Squalus acanthias). Antagonistic effects of ATP and urea on oxygen affinity of an elasmobranch hemoglobin

The amino-acid sequence of the hemoglobin of the Dogfish Shark (Squalus acanthias) is presented. The alpha-chains consist of 141 residues and show a Thr/Ser ambiguity at position 3. The beta-chains consist of 142 residues and evidently have no D-helix; they show an Asn/Tyr ambiguity at position 104. Both chains have free N-terminal amino acids. The phylogenetic distance from the human alpha- and beta-chains is indicated by 49.3% and 56.2% amino-acid exchanges. The primary structure is discussed in relation to the oxygen-binding properties of elasmobranch hemoglobin, particularly as regards the antagonistic effects of urea and ATP, and the effects of proton concentration (the alkaline and acid Bohr effects, and the Root effect).     

A "living fossil" sequence: primary structure of the coelacanth (Latimeria chalumnae) hemoglobin--evolutionary and functional aspects

The coelacanth (Latimeria chalumnae, Actinistia) has a single hemoglobin component. The primary structures of the alpha- and beta-chains are presented. They could be separated by reversed-phase HPLC. Peptides obtained by tryptic digestion of the native and oxidized chains were isolated by reversed-phase HPLC and sequenced in liquid and gas-phase sequenators. The alignment was achieved by employing the N-terminal sequences of the native chains and those of a beta-chain cyanogen bromide peptide as well as fragments obtained by acid hydrolysis. The Latimeria alpha-chains consist of 142 amino-acid residues, due to a fish-specific insertion between positions 46 and 47, whereas the beta-chains are of normal length (146 residues). Latimeria alpha- and beta-chains share 72 (51.1%) and 70 (47.9%) identical residues with human hemoglobin, respectively. Numerous heme contacts and positions involved in subunit interface contacts are replaced. The most interesting of them were studied by molecular modeling. The loss of an alpha 1/beta 2-contact by the exchanges alpha 92(FG4)Arg----Leu and beta 43(CD2)Glu----Lys might be responsible for the easy dissociation of the tetrameric hemoglobin molecule. A comparison of the residues replaced in contact positions with fishes and amphibians revealed the highest number of matches between Latimeria and tadpoles. The same result was obtained by the evaluation of other regions relevant for structure and function of the molecule, like exon-intron boundary regions, phosphate binding sites and salt bridges responsible for the Bohr effect.     

The first sequenced normal hemoglobin lacking histidine in position 146 of the beta-chains. The primary structures of the major and minor hemoglobin components of the great crested newt (Triturus cristatus, Urodela, Amphibia)

The hemoglobin of the Great Crested Newt (Triturus cristatus), an animal maintaining the gas exchange to about 85% through the skin, consists of a major (HbM = 65%) and a minor (Hbm = 35%) component. The primary structures of the four chains are presented. They could be separated by reversed-phase HPLC and were cleaved with trypsin and additionally by acid hydrolysis. Both the native chains and their peptides were sequenced by liquid and gas phase sequenators. At the N-terminus the alpha M-chains are by one amino-acid residue longer and the beta M-chains by one residue shorter, resulting in a chain length of 142 and 145, respectively. The alpha m-chains are of normal length whereas in the beta m-chains the C-terminal histidine in position 146 is missing. Both alpha-chains differ by 50 residues (35.2%) and the beta-chains by 63 (43.2%). The alpha-chains were compared with those of other salamandroid hemoglobins. The difference to human hemoglobin is marked by 61 (43.3%) amino-acid substitutions in both alpha-chains and by 78 (53.4%) in both beta-chains. Numerous heme contacts and positions involved in the subunit interface are affected by replacements. The most interesting of them were studied by molecular modeling. The importance of the missing beta m-146(HC3)His and of the substitution of several amino-acid residues involved in the binding of organic phosphates is discussed with respect to the reduced Bohr effect of Triturus cristatus hemoglobin.     

Effects of anions on the molecular basis of the Bohr effect of hemoglobin

High-resolution 1H-NMR spectroscopy has been used to investigate the molecular basis of the Bohr effect in human normal adult hemoglobin in the presence of anions which serve as heterotropic effectors, i.e., Cl-, Pi, and 2,3-diphosphoglycerate. The individual H+ equilibria of 22-26 histidyl residues of hemoglobin in both deoxy and carbonmonoxy forms have been measured under buffer conditions chosen to demonstrate the effects of anion binding. The results indicate that beta 2His residues are binding sites for Cl- and Pi in both deoxy and carbonmonoxy forms, and that the affinity of this site for these anions is greater in the deoxy form. Recently assigned, the resonance of beta 146His does not show evidence of involvement in anion binding. The results also indicate that the binding of 2,3-diphosphoglycerate at the central cavity between the two beta-chains in deoxyhemoglobin involves the beta 2His residues, and that the 2,3-diphosphoglycerate-binding site in carbonmonoxyhemoglobin may remain similar to that in deoxyhemoglobin. The interactions of Cl-, Pi and 2,3-diphosphoglycerate also result in changes in the pK values for other surface histidyl residues which vary in both magnitude and direction. The array of pK changes is specific for the interaction of each effector. The participation of beta 2His in the Bohr effect demonstrates that this residue can release or capture protons, depending on its protonation properties and its linkage to anion binding, and therefore provides an excellent illustration of the variable roles of a given amino acid. Although beta 146His does not bind anions, its contributions to the Bohr effect are substantially affected by the presence of anions. These results demonstrate that long-range electrostatic and/or conformational effects of anions binding play significant roles in the molecular basis of the Bohr effect of hemoglobin.     

Primary structure and oxygen-binding properties of the hemoglobin from the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta). Evidence for phylogenetic isolation.

The primary structures of the alpha- and beta-hemoglobin chains of the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta) are presented. Chain separation was performed by carboxymethyl-cellulose chromatography. The peptides, obtained by tryptic digestion of the oxidized chains, were prefractionated by gel chromatography and isolated by reversed-phase HPLC. For sequence analysis gas and liquid phase sequencers were employed. The tenrec hemoglobin consists of one alpha- and two beta-chains the latter occurring in a 1:1 ratio and differing in beta 16 Gly/Cys and beta 118 Phe/Leu. Two external cysteine residues at beta 16 and beta 52 cause reversible polymerization to octamers and most likely irreversible formation of higher polymers. A comparison of the whole chains and certain positions of tenrec hemoglobin with those of Insectivora sensu strictu, Scandentia and Proto- and Metatheria corroborates a long and independent evolution of tenrec and its phylogenetic isolation from the Insectivora s.str. (hedgehog, musk shrew and mole). Replacements at positions involved in heme and subunit interface contacts are discussed. Compared to human hemoglobin the tenrec pigment shows a low intrinsic oxygen affinity as well as lower chloride and temperature sensitivities, a reduced Bohr effect and a strong response to 2,3-DPG. The possible adaptive significance of these properties is discussed in relation to the large diurnal body temperature variations seen in tenrecs.     

The primary structure of the pallid bat (Antrozous pallidus, Chiroptera) hemoglobin

The complete primary structure of the hemoglobin from the Pallid Bat (Antrozous pallidus, Microchiroptera) is presented. This hemoglobin consists of two components with identical amino-acid sequences, differing, however, in the N-terminus which is formylated in 12.5% of the beta-chains. The alpha- and beta-chains were separated by reversed phase high performance liquid chromatography. The sequences of both chains were established by automatic Edman degradation with the film technique or gas phase method using the native chains and the tryptic peptides. The formylation of a part of the N-terminal peptide of the beta-chains was determined by mass spectrometric examination. Compared to the corresponding human chains we found 14 substitutions in the alpha-chains and 21 in the beta-chains. One substitution in the alpha-chains and three in the beta-chains are involved in alpha 1/beta 1-contacts. Among these the exchange beta 123(H1)Thr----Cys is unusual because cysteine was so far not found in this position of mammalian beta-chains. Compared to the hemoglobin of Myotis velifer, another representative of the family Vespertilionidae, 5 residues are replaced in the alpha-chains and 18 in the beta-chains.     

Carnivora: the primary structure of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae)

The primary structure of the alpha- and beta-chains of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae) is described. The chains were separated directly from hemoglobin by RP-HPLC. After tryptic digestion of the chains, the peptides were separated by RP-HPLC. Amino acid sequences were determined by Edman degradation in liquid and gas-phase sequencers. The alignment of the tryptic peptides was made by homology with human and other Carnivora hemoglobins. Paguma and human hemoglobin differ with respect to 23 amino-acid residues. Some of these amino-acid substitutions, which occur in both the alpha- and beta-chains, occur at contact sites between the subunits, and at the binding sites of heme and of organic phosphate, as well as involving residues responsible for the alkaline Bohr effect.     

The primary structure of the hemoglobin of the mole rat (Spalax ehrenbergi, rodentia, chromosome species 60)

Mole rat (Spalax ehrenbergi) hemoglobin consists of only one component. The complete amino-acid sequence of the alpha- and beta-chains of the species with the diploid chromosome number of 60 is presented. Following chain separation by chromatography on carboxymethyl cellulose CM-52, the primary structures were established by automatic Edman degradation on the chains, on the tryptic peptides, and on a peptide obtained by acid hydrolysis of the Asp-Pro bond in beta-chains. The alignment of the peptides was performed by homology with human alpha- and beta-chains. The comparison showed an exchange of 23 residues in the alpha-chains and 26 in the beta-chains. One substitution in the beta-chains concerns the surrounding of the heme. We found two exchanges in each chain in the alpha 1 beta 1-subunit interface and one in the beta-chain alpha 1 beta 2-contact points. Though all binding sites for 2,3-bisphosphoglycerate are unchanged, the mole rat blood has a high oxygen affinity as a part of adaptation to subterranean life under hypoxia and hypercapnia. A comparison of the sequence with known X-ray models of hemoglobins may give an interpretation of this fact. The primary structure of the mole rat hemoglobin shows more similarities with surface rodents, than with the mole, another small mammal, adapted to hypoxia in subterranean tunnels. The adaptation to hypoxia in mole rat and mole must be due to different mechanisms.     

Carnivora: the primary structure of the giant otter (Pteronura brasiliensis, Mustelidae) hemoglobin

The hemoglobin of the Giant Otter (Pteronura brasiliensis, Carnivora) contains only one component. The complete primary structures of the alpha- and beta-chains are presented. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid- and gas-phase Edman degradation of the chains and their tryptic peptides. The alpha-chains show 18 and the beta-chains 12 exchanges compared with human alpha- and beta-chains, respectively. In the alpha-chains, two substitutions involve alpha 1/beta 1-contacts and one a heme-contact. In the beta-chains one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact are exchanged. The alpha- and beta-chains of the Giant Otter are compared to those of the Common Otter and other Carnivora hemoglobins.     

Carnivora: the primary structure of the common otter (Lutra lutra, Mustelidae) hemoglobin

The hemoglobin of the Common Otter (Lutra lutra, Carnivora) contains only one component. The complete primary structures of the alpha- and beta-chains are presented. They were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas-phase Edman degradation of the chains and their tryptic peptides. The alpha-chains show 18 and the beta-chains 13 substitutions compared to human alpha- and beta-chains, respectively. In the alpha-chains one heme- and two alpha 1/beta 1-contacts are exchanged. In the beta-chains the replacements involve one heme-, one alpha 1/beta 1-, and one alpha 1/beta 2-contact. The alpha- and beta-chains of the Common Otter are compared to those of other Carnivora hemoglobins. The unexpected low number of substitutions between Common Otter hemoglobin and that of Lesser Panda as well as of Harbor Seal is discussed.     

Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain

The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.     

The primary structure and functional properties of the hemoglobins of a ground squirrel (Spermophilus townsendii, Rodentia)

The hemoglobin of the ground squirrel Spermophilus townsendii consists of two components which are present in a ratio of ca. 2:1. The two hemoglobins have identical alpha-chains, but differ in their beta-chains. We present the primary structures of the alpha- and the two beta-globin chains. Following chain separation by chromatography on carboxymethyl-cellulose CM-52, the amino-acid sequences were established by automatic Edman degradation of the globin chains and the tryptic peptides, as well as of a peptide obtained by acid hydrolysis of the Asp-Pro bond of the beta-chains. The two beta-chains differ by only one amino-acid residue, Ala being present in the main and Asp in the minor component in position 58 (E2). The comparison with human hemoglobin showed only 14 exchanges in the alpha-chains but 33 in the beta-chains. Whereas no contact positions are affected in the alpha-chains, we found four such substitutions in the beta-chains, including one heme contact, two alpha 1/beta 1-contacts, and one alpha 1/beta 2-contact. It seems however, that the substitution found in the beta-chains has no effect on the oxygen affinity.     

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.     

Amino-acid sequence of gayal hemoglobin (Bos gaurus frontalis, Bovidae)

The amino-acid sequences of the alpha- and beta-chains of gayal hemoglobin have been determined and compared with those of bovine and yak hemoglobins. The gayal alpha-chain differs from the alpha-chains of bovine by 3 amino-acid residues and from yak I alpha- and II alpha-hemoglobins by 4 and 2 residues, respectively. The gayal beta-chain differs from bovine beta A- and beta B-chains by 3 and 4 residues, respectively and from yak beta-chains by 2 residues.     

Carnivora: primary structure of the hemoglobins from ratel (Mellivora capensis)

The erythrocytes of adult ratel contain two hemoglobin components, with two alpha- and one beta-chains. In this paper, their complete amino acid sequences are presented. The two alpha-chains differ in one residue at position 34 (Ala----Val) only. The primary structure of the chains was determined by sequencing the N-terminal regions (45 steps) and the tryptic peptides after their isolation from the digests by reversed-phase high-performance liquid chromatography. The alignment of these peptides was deduced from homology with other carnivora globins. The alpha-chains show 21 and the beta-chains 11 exchanges compared with human globin chains. In the alpha-chains, one heme- and two alpha 1/beta 1 contacts are exchanged. In the beta-chains there are three exchanges which involve one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact. Between the ratel hemoglobin and those of carnivora a high degree of homology was found.     

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.     

The primary structure of the hemoglobin from the Australian ghost bat (Macroderma gigas, Microchiroptera).

The Australian ghost bat (Macroderma gigas, Microchiroptera) has two hemoglobin components in the ratio 3:2. They share identical beta-chains and differ by three replacements in the alpha-chains. The primary structures of all three chains are presented. They could be separated by high-performance liquid chromatography. The sequences were determined by automatic liquid and gas phase Edman degradation of the chains and their tryptic peptides. The two alpha-chains show 18 and 19 and the beta-chains 15 exchanges compared to human alpha- and beta-chains, respectively. The divergent evolution of Macroderma gigas and Megaderma lyra, two representatives of the family Megadermatidae, is discussed. An influence of replacements at functionally important positions on the hemoglobin oxygen affinity seems unlikely.     

The primary structure of the hemoglobin of the Rock-Hopper penguin (Eudyptes crestatus, Sphenisciformes)

The blood of the Rock-Hopper Penguin contains only one hemoglobin component, corresponding to the Hb A of other birds. The primary structures of the alpha- and beta-chains are presented. The chains were separated by high-performance liquid chromatography and cleaved either enzymatically (alpha) or both enzymatically and chemically (beta). Both the native chains and their peptides were sequenced using liquid and gas phase sequenators. The peptides were aligned using their homology to the sequence of human hemoglobin and other bird hemoglobins. As compared to human hemoglobin, 44 amino-acid replacements are found in the alpha-chains (68% homology) and 47 in the beta-chains (67.8% homology). These exchanges involve seven alpha 1/beta 1 and one alpha 1/beta 2 contact in the alpha-chains, whereas in the beta-chains eight alpha 1/beta 1, one alpha 1/beta 2 and one hem contact are substituted. The influence of these replacements on the structure-function relationships in hemoglobin, as well as their importance for the diving ability of penguins, are discussed.     

The primary structure of a mouse-eared bat (Myotis velifer, Chiroptera) hemoglobin

The hemoglobin of the Mouse-Eared Bat Myotis velifer consists of one component. We present the primary structures of the alpha- and beta-globin chains which have been separated by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman-degradation with the film technic or the gas phase method, using the native chains and the tryptic peptides, as well as the C-terminal prolyl-peptides obtained by acid hydrolysis of the Asp-Pro-bonds. Compared to the corresponding human chains we found only 13 substitutions in the alpha-chains, but 27 in the beta-chains. The amino-acid residues substituted in the alpha-chains are not involved in any contacts, whereas in the beta-chains, one exchange involves a heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts, the latter [beta 43(CD2)-Glu----Thr] brings for the first time threonine in this position of the beta-chains. Comparison with the Egyptian Fruit Bat (Rousettus aegyptiacus) shows 12 and 25 substitutions in the alpha- and beta-chains, respectively, suggesting a large phylogenetic distance between Micro- and Megachiroptera. We consider this primary structure as a contribution towards solving the problem of the origin of bats and their relation to primates.