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.     

Carnivora: the primary structure of the Pacific Walrus (Odobenus rosmarus divergens, Pinnipedia) hemoglobin

The primary structure of the alpha- and beta-chains of the hemoglobin from the Pacific Walrus (Odobenus rosmarus divergens, Pinnipedia) is presented. Sequence analysis revealed only one hemoglobin component whereas two bands were found in polyacrylamide gel electrophoresis. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid- and gas-phase sequencing of the chains and their tryptic peptides. The alpha-chains show 20 and the beta-chains 12 exchanges compared to the corresponding human chains. In the alpha-chains one heme- and two alpha 1/beta 1-contacts were exchanged whereas in the beta-chains one alpha 1/beta 1-, one alpha 1/beta 2-and one heme-contact are substituted. Compared to Harbour Seal (Phoca vitulina) the Walrus hemoglobin shows 9 amino-acid replacements in the alpha-chains and 5 in the beta-chains. The relation between Pinnipedia and Arctoidea is discussed.     

The primary structure of the hemoglobin of the Indian false vampire (Megaderma lyra, Microchiroptera)

The hemoglobin of the Indian false vampire Megaderma lyra contains only one component. In this paper, we are presenting its primary structure. 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, as well as of the prolyl-peptides obtained by acid hydrolysis of the Asp-Pro bond in the alpha- and beta-chains. The alpha-chains show 23 and the beta-chains 20 exchanges compared with the human alpha- and beta-chains, respectively. In the alpha-chains, three exchanges involved alpha 1/beta 1 contacts. In the beta-chains one heme-and three alpha 1/beta 1 contacts are exchanged. The functional and systematic aspects of these replacements are discussed.     

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.     

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.     

The primary structure of the hemoglobin of the Brazilian manatee (Trichechus inunguis, Sirenia)

The hemoglobin of the Brazilian Manatee (Trichechus inunguis, Sirenia) consists of one component. We present the primary structures of the alpha- and beta-chains which have been separated by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by automatic Edman degradation with the film technique, using the native chains, tryptic peptides and the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond of the alpha-chains. Compared to the corresponding human chains we found 27 substitutions in the alpha- as well as in the beta-chains. Three heme contacts and four alpha 1/beta 1 contacts between the subunits are affected by exchanges. The hemoglobin of Trichechus inunguis is compared with those of Elephas maximus, Loxodonta africana, and Procavia habessinica and the monophyletic origin of the superorder Paenungulata is discussed.     

Carnivora: the primary structure of the hemoglobin from the silver fox (Vulpes vulpes var., Canidae)

The primary structure determination of the hemoglobin alpha- and beta-chains from the silver fox (Vulpes vulpes var., Canidae) is described. The separation of the chains could be achieved directly from the hemoglobin by RP-HPLC as well as by column chromatography of the globin using carboxymethyl-cellulose. Following tryptic digestion of the chains, the peptides were isolated by RP-HPLC. Amino-acid sequences were determined by Edman degradation in liquid and gas phase sequencers. The peptides could be aligned by homology with human and other Carnivora hemoglobins. Compared to human hemoglobin the alpha- and beta-chains of the silver fox exhibit 24 and 13 amino-acid exchanges, respectively. They differ by one alpha- and two beta-chain replacements from the domestic dog and the coyote. The substitutions affecting contact positions are discussed.     

The primary structure of the hemoglobin of an Indian flying fox (Cynopterus sphinx, Megachiroptera)

The hemoglobin of the Indian flying fox Cynopterus sphinx contains only one component. In this work, we are presenting its primary structure. 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, as well as of the peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The alpha-chains show 14 and the beta-chains 19 exchanges compared with the human alpha- and beta-chains, respectively. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1 contact. In the beta-chains one heme contact, three alpha 1/beta 1- and one alpha 1/beta 2-contacts are exchanged. The functional and evolutionary aspects of these findings are discussed.     

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.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

The primary structure of the hemoglobin from the aardwolf (Proteles cristatus, Hyaenidae)

The hemoglobin of the aardwolf (Proteles cristatus) contains only one component. In this paper, we are presenting its primary structure. 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- as well as the beta-chains show 20 exchanges compared with the corresponding human chains. The difference to the masked palm civet (Paguma larvata) and the spotted hyaena (Crocuta crocuta) is marked by 16 and 4 replacements in the alpha-chains and by 10 and 1 in the beta-chains, thus supporting the hyaenid character of the aardwolf. The exchanges at contact positions are shared by other carnivoran hemoglobins.     

The primary structure of the hemoglobin of the European marmot (Marmota marmota marmota, Rodentia)

The hemoglobin of the European marmot Marmota marmota marmota has been found to consist of only one component. In this work, we are presenting its primary structure. The globin chains have been separated by high performance liquid chromatography and the sequences have been determined by automated Edman degradation of the chains and their tryptic peptides, as well as of the peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. In the alpha-chains we have found 13 and in the beta-chains 34 exchanges compared with the human alpha- and beta-chains, respectively. The amino acids which are substituted in the alpha-chains are not involved in any contacts, whereas in the beta-chains, one exchange involves a heme contact, two alpha 1/beta 1- and one alpha 1/beta 2-contacts. The functional and evolutionary aspects of these findings are discussed.     

Carnivora: primary structure of the hemoglobin from the spotted hyena (Crocuta crocuta, Hyenidae)

The primary structure of the alpha- and beta-chains of hemoglobin from spotted hyena (Crocuta crocuta, Hyenidae) is presented. The structure-function relationship is discussed. The separation of the chains directly from hemoglobin was performed by RP-HPLC. After tryptic digestion of the chains, the peptides were isolated 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. The hemoglobin from spotted hyena (Crocuta crocuta) exhibits in its alpha- and beta-chains 22 and 20 exchanges, respectively, compared to human hemoglobin. In the alpha-chains, two alpha 1 beta 1-contacts are exchanged. In the beta-chains five exchanges involve one alpha 1 beta 1-contact, one alpha 1 beta 2-contact, one heme contact, and two 2,3-DPG-binding sites.     

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 pale-throated three-toed sloth (Bradypus tridactylus, Xenarthra) hemoglobin

The hemoglobin of the Pale-Throated Three-Toed Sloth (Bradypus tridactylus, Xenarthra) was separated into two components (ratio 4:1) with identical amino-acid analyses for the alpha- and beta-chains. The primary structures of both chains from the major component are given. They could be isolated by chromatography on carboxymethyl cellulose CM-52. The sequences have been determined by automatic Edman degradation of the native chains and their tryptic peptides. The comparison with human hemoglobin showed 27 substitutions in the alpha-chains and 33 in the beta-chains. In the alpha-chains one amino-acid exchange involves an alpha 1/beta 1-contact. In the beta-chains two heme- and four alpha 1/beta 1-contacts are substituted. The hemoglobin of the Sloth is compared to that of the Nine-Banded Armadillo (Dasypus novemcinctus), another representative of the order Xenerthra.     

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.     

The primary structure of hemoglobins of the rock hyrax (Procavia habessinica, Hyracoidea): insertion of glutamine in the alpha chains

The chromatography of the hemoglobin of the rock hyrax (Procavia habessinica) gives two components (73% HbI and 27% HbII). The amino-acid analysis and the sequences of the globin chains elucidated with the phenylthiohydantoin method, did not show any differences between the alpha I and alpha II or beta I and beta II chains, respectively. The different chromatographical behaviour cannot be explained. After chain separation by chromatography on CM-52 cellulose, all four primary structures were elucidated automatically in a sequenator on the chains and the tryptic peptides. In 20% of the beta I chains the N-terminal valine was blocked by acetyl. The alignment was performed by homology with the chains of human adult hemoglobin. The alpha chain of the rock hyrax has 142 amino-acid residues, i.e. one residue more than normal mammalian alpha chains, caused by an insertion of glutamine in the GH region supposed between positions 115 and 116. A comparison of human and hyrax hemoglobins shows an exchange of 21 amino-acid residues in the alpha chains and of 24 in the beta chains. Some substitutions in alpha 1 beta 1 contacts and in the surrounding of the heme are not supposed to effect the function of the hemoglobin. The phylogenetic relationship between the rock hyrax and the Indian elephant (Elephas maximus) on the one hand and with some Perissodactyla on the other, is discussed. Up to now the exchanges of alpha 110(G17)Ala leads to Ser and beta 56(D7)Gly leads to His have only been found in hyrax and elephant. This indicates a certain relationship between Hyracoidea and Proboscidea.     

The primary structure of the mandrill (Mandrillus sphinx, Primates) hemoglobin

The complete primary structure of the hemoglobin from the Mandrill (Mandrillus sphinx, Primates) is presented. This hemoglobin comprises two components in approximately equal amounts (HB I and Hb II). The alpha-chains differ in positions 5 (A3) and 9 (A7) having Ala and Asn in the alpha I-chains and Asp and His in the alpha II-chains. The beta-chains are identical. The components could be separated by DEAE-Sephacel chromatography. The globin chains were obtained by carboxymethylcellulose chromatography or high-performance liquid chromatography. The sequences were established by automatic liquid or gas phase Edman degradation of the chains and their tryptic peptides. The alpha-chains show 9 and 11 and the beta-chains 8 exchanges compared with the corresponding human chains, respectively. In the beta-chains one alpha 1/beta 1- and one alpha 1/beta 2-contact is substituted. A comparison of the primary structures of the Mandrill hemoglobin chains with those of other species of the Cercopithecidae family shows that Mandrillus sphinx should be placed between Cercopithecus and Macaca on one side and Papio, Theropithecus and Cercocebus on the other.     

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.     

Carnivora: the primary structure of Weddell Seal (Leptonychotes weddelli, Pinnipedia) hemoglobin

The hemoglobin of Weddell Seal (Leptonychotes weddelli, Pinnipedia) comprises two components with identical beta-chains. The alpha-chains differ in positions 15 (Gly/Asp) and 57 (Ala/Thr). We present the primary structure of the chains which have been separated by reversed-phase high-performance liquid chromatography. The sequences have been determined by automatic Edman-degradation with the film-technique or the gas-phase method, using the native chains and the tryptic peptides of the oxidized chains. Compared to the corresponding human chains we found 22 substitutions in the alpha-chains and 14 in the beta-chains. In the alpha-chains exchanges involve one heme- and three alpha 1/beta 1-contacts. In the beta-chains one heme contact, one alpha 1/beta 1- and one alpha 1/beta 2-contacts are substituted. The sequences are compared to those of other Pinnipedia and Arctoidea hemoglobins.