Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla)

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.     

The primary structure of the hemoglobins of the adult jaguar (Panthera onco, Carnivora)

The primary structure of the hemoglobins from Jaguar (Panthera onco) are presented. Electrophoretic separations without and with a dissociating agent revealed the presence of two hemoglobin components, alpha 2 beta I2 and alpha 2 beta II2. The separation of the hemoglobin components was achieved by ion-exchange chromatography. The globin chains were separated by ion-exchange chromatography and also by reversed phase HPLC. The amino-acid sequences of the native chains and peptides were determined by liquid-phase and gas-phase sequencing. N-Acetylserine was detected by FAB-mass spectroscopy as N-terminal group of the beta I chain. The sequences are compared with that of human hemoglobin (Hb A).     

Perissodactyla: the primary structure of hemoglobins from the lowland tapir (Tapirus terrestris): glutamic acid in position 2 of the beta chains

The hemoglobins from a lowland tapir (Tapirus terrestris) were analysed and the complete primary structure is described. The globin chains were separated on CM cellulose column in 8M urea and the amino-acid sequences were determined in the liquid phase sequenator. The results show that globin consists of two alpha chains (alpha I and alpha II) and beta major and beta minor components. The alpha chains differ only at one position: alpha I contains aspartic acid and alpha II glycine. The beta chains are heterogeneous: aspartic and glutamic acid were found at position beta 21 and beta 73 of the beta major components and asparagine and serine at position beta 139. In the beta minor components four positions were found with more than one amino acid, namely beta 2, beta 4, beta 6 and beta 56. The sequences are compared with those of man, horse and rhinoceros. Four residues of horse methemoglobin, which are involved in the alpha 1 beta 1 contacts are substituted in tapir hemoglobins. In the alpha chains: alpha 107(G14)Ser----Val, alpha 111-(G18) Val----Leu, alpha 115(GH3) Asn----Asp or Gly; in the beta chains: beta 116(G18) Arg----Gln. The amino acid at beta 2 of the major components is glutamic acid while glutamine and histidine are found in the minor components. Although glutamic acid, a binding site for ATP, does not interact with 2,3-bisphosphoglycerate, glutamine and histidine in the minor components are responsible for the slight effect of 2,3-bisphosphoglycerate on tapir hemoglobin.     

Gene expression in an interspecific hybrid: Analysis of hemoglobins in donkey,horse, and mule by peptide mapping

The fast and slow components of horse, donkey, and mule hemoglobin were analyzed by mapping of tryptic peptides. The two horse components share a common beta chain but differ in the alpha chain. Donkey hemoglobin differs from the slow horse component by replacement of histidine by asparagine in position 20 and replacement of a glycine residue by alanine in peptide V. Mapping of mule hemoglobins indicates that (1) the slow mule component contains the alpha and the beta chains of the slow horse component and the alpha and beta chains of the donkey, and (2) the fast mule component contains the alpha chain of the fast horse component and the beta chains of both the horse and the donkey. It is concluded that all six theoretically possible tetrameric combinations of the polypeptide chains encoded by the horse and donkey parental genes are formed in the mule.     

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.     

Primary structure of alpha and beta chains from the major hemoglobin component of the magpie goose (Anseranas semipalmata, Anatidae)

The amino acid sequence of the alpha and beta chains from the major hemoglobin component (HbA) of Australian Magpie Goose (Anseranas semipalmata) is given. The minor component with the alpha D chains was detected, but only found in low concentrations. By homologous comparison, Greylag Goose hemoglobin (Anser anser) and Australian Magpie Goose alpha chains differ by 13 amino acids or 17 nucleotide (4 two point mutations) exchanges, beta chains by 6 exchanges. Seven alpha 1 beta 1 contacts are modified by substitutions in positions alpha 30-(B11)Glu leads to Gln, alpha 34(B15)Thr leads to Gln, alpha 35(B16)-Ala leads to Thr, alpha 36(B17)Tyr leads to Phe, beta 55(D6)Leu leads to Ile, beta 119(GH2)Ala leads to Ser and beta 125(H3)Glu leads to Asp. Further, one alpha 1 beta 2 contact point was changed in beta 39(C5)Gln leads to Glu. Mutation in this position, except in two abnormal human hemoglobins, was not found in any species. Amino acid exchanges between hemoglobin of Australian Magpie Goose and other birds are discussed.     

The primary structures of the major and minor hemoglobin-components of adult Andean goose (Chloephaga melanoptera, Anatidae): the mutation Leu----Ser in position 55 of the beta-chains

The primary structures of the hemoglobin components Hb A and Hb D of the adult Andean Goose (Chloephaga melanoptera) are presented. The globin chains were separated on CM-Cellulose in 8M urea buffer. The amino-acid sequences were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid- and gas-phase sequenators. The sequences are aligned with those of Greylag Goose (Anser anser) as a biological reference and other sequences of birds. A detailed evaluation of all residues of Andean Goose hemoglobins on the basis of the 12000 known avian globin sequences leads to a molecular pattern for high-altitude respiration of geese. The replacement of functional and structural importance is the unique occurrence of the residue beta 55 Leu----Ser (all other exchanges are functionally neutral), interrupting the same alpha 1 beta 1-interface contact (alpha 119-beta 55) that accounts for high-altitude respiration of the Barheaded Goose (Anser indicus); there the mutation is found on alpha A 119. Loosening the constraints of this interface must be interpreted as a destabilization of the low-affinity T-structure in favour of the high-affinity R-structure. The structural and functional significance of this interface for the molecular biology of high-altitude respiration of the Andean Goose and Barheaded Goose is discussed. Since Hb A consists of alpha A2 beta 2 and Hb D consists of alpha D2 beta 2 the mutation occurring in blood of the Andean Goose affects both hemoglobins whereas in the case of the Barheaded Goose only Hb A is affected. These results show that Hb D can be considered a biological reserve to enlarge situatively the normal hemoglobin function. A general molecular pattern for permanent (selective advantage of high intrinsic oxygen affinity) and transitory (selective advantage of graded oxygen affinities) adaptation to hypoxia is discussed. A survey on the sequence homology of the globin chains of geese (Anserinae) and ducks (Anatinae) is given.     

Deoxygenation-linked association of a tetrameric component of chicken hemoglobin.

Deoxygenation-dependent association of hemoglobin tetramers appears to be widespread among amphibians, reptiles, and possibly all or most birds. The evidence for this conclusion depends largely on oxygen equilibria of whole blood which have Hill coefficients that reach values as high as 5-7 at 80-90% oxygenation. Computer simulation of the sedimentation velocity behavior of the major components A and D of chicken hemoglobin shows that component D but not A self-associates to form dimers of tetramers. The gradient profiles at pH 7.5 were satisfactorily fitted with an association constant of 1.26 x 10(4) M-1 and sedimentation coefficients of 4.63 and 7.35 S for tetramer and (tetramer)2, respectively. Since components A and D share common beta chains we conclude that tetramer-tetramer contacts must depend on surface residues of the alpha chains. Comparison of the amino acid sequences of the alpha D and alpha A chains of the hemoglobins from 12 avian species ranging from sparrow to ostrich shows that 20 residues are conserved in the alpha D chains but not in the alpha A chains. Nine of these (45%) are clustered between positions E20 and FG2. Four of the latter, Lys71 (E20), Asn75 (EF4), Gln78 (EF7), and Glu82 (F3) are conserved in all alpha D chains even though they do not appear to participate in intratetramer contacts. Molecular modeling indicates that residues Lys71, Gln78, and Glu82 of the alpha chain are strong candidates for the primary tetramer-tetramer contacts.     

The expression of alpha D-chains in the hemoglobin of adult ostrich (Struthio camelus) and American rhea (Rhea americana). The different evolution of adult bird alpha A-, alpha D- and beta-chains

The hemoglobin of adult American rhea (Rhea americana) and ostrich (Struthio camelus) contains two components identified to be HbA (alpha 2A beta 2) and HbD (alpha 2D beta 2). The amino-acid sequence of alpha D-chains from HbD of adult American rhea and ostrich has been determined. The sequence was studied by Edman degradation of tryptic peptides and chemical cleavage products in a liquid phase sequencer. By homologous comparison with pheasant HbD (Phasianus colchicus colchicus), the alpha D-chains of American rhea differ by 28 amino-acid exchanges, the alpha D-chains of ostrich by 23 residues. These differences are higher than those observed for alpha A- as well as for beta-chains of HbA from the same species. The ratio of amino-acid exchanges for beta:alpha A:alpha D for American rhea and ostrich is found to be 1:5.5:6.5. At present the reason for the differences in evolution rates for the beta-, alpha A- and alpha D-chains of bird hemoglobins is still unclear.     

Primary structure, biochemical and physiological aspects of hemoglobin from South American lungfish (Lepidosiren paradoxus, Dipnoi)

The South American Lungfish has only one hemoglobin component. The complete amino-acid sequence of this hemoglobin is presented. A large quantity of carbonate dehydratase from the lungfish erythrocytes was also isolated. The carboxymethylated chains, obtained by separation of globin on DEAE-Sephacel, were submitted to tryptic digestion and chemical cleavage. The isolation of tryptic peptides was achieved either by Dowex-50 chromatography or by high performance liquid chromatography. The alignment of peptides was performed by homology with the previously established sequences of the carp and goldfish hemoglobins. The overlapping peptides confirmed this sequence. The alpha chains have 143 residues, the beta chains 147. The relation between the primary structure and the physiological properties of lungfish hemoglobin are discussed.     

High-altitude respiration of birds. Structural adaptations in the major and minor hemoglobin components of adult Rüppell's Griffon (Gyps rueppellii, Aegypiinae): a new molecular pattern for hypoxic tolerance

The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted.     

Homeothermic fish and hemoglobin: primary structure of the hemoglobin from bluefin tuna (Thunnus thynnus, Scromboidei)

Some fish are warm-bodied, e.g. the bluefin tuna (Thunnus thynnus), which has a muscle temperature 12-17 degrees C higher than its environment. This endothermy is achieved by aerobic metabolism and conserved by means of a heat-exchanger system. The hemoglobins of bluefin tuna are adapted to these conditions by their endothermic oxygenation, thus contributing to the preservation of the body energy. This is a new and so far unique property of tuna hemoglobin. The primary structure of the alpha and beta chains of bluefin tuna hemoglobins is presented. The sequence was determined after enzymatic and chemical cleavages of the chains and sequencing of the peptides in gas- and liquid-phase sequencers. The alpha chains consists of 143 residues and are N-terminally acetylated. The beta chains have 146 amino acids and show two ambiguities at positions 140 and 142. The alpha chains differ from the human alpha chains in 65 amino-acid residues, the beta chains in 76. The hemoglobins of bluefin tuna, carp and man are compared and their different physiological properties are discussed in relation to the sequence data. From the primary structure of tuna hemoglobins, it is possible to propose a molecular basis for their peculiar endothermic transition from the T to the R structure.     

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 sperm whale hemoglobin (Physeter catodon, cetacea)

The complete primary structure of the two major hemoglobin components of sperm whale (Physeter catodon) is presented. The major components A and B account for 55% and 40% respectively whereas the minor component constitutes for 5% of the total hemoglobin. The globin chains were separated on CM-Cellulose in 8M urea buffer. The sequence was determined by automatic Edman degradation of tryptic and hydrolytic peptides in a liquid phase sequencer. Alignment of the sequence with human hemoglobin shows 22 exchanges each for the alpha I and alpha II and 21 exchanges for the beta I and beta II chains. Within the two beta-chains three differences have been located, beta NA2 His/Gln, beta A2 Gly/Ala and beta A8 Leu/Val. The two alpha-chains are characterized by heterogeneities at position alpha A8 Val/Ile or Ala/Ile (ratio of the phenylthiohydantoin derivatives of the amino acids 1:1) and alpha AB1 Asn/Ser (ratio of the phenylthiohydantoin derivatives of the amino acids 6:4). The role of these exchanges in modulating oxygen affinity is discussed.     

Unique features of the hemoglobin system of the Antarctic notothenioid fish Gobionotothen gibberifrons.

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.     

The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.      

Carnivora: the primary structure of the alpha-chains of ferret (Mustela putorius furo, Mustelidae) hemoglobins

Ferret erythrocytes contain two hemoglobins differing only by their alpha-chains. The primary structure of the common beta-chain has been previously described; the complete sequence of the two alpha-chains are reported in this paper. The globin chains were separated by ion-exchange chromatography; the alpha-chains (42 steps), their tryptic peptides as well as the prolyl-peptides were subjected to automatic liquid- and gas-phase Edman degradation. The two alpha-chains are very similar, differing at only one position (Asp15----Gly15). Comparison with human hemoglobin alpha-chain shows 16 and 17 exchanges, for alpha 1 and alpha II chains, respectively; two substitutions involve alpha 1/beta 1 contacts and one the heme contacts. A high degree of homology was noted when the alpha-chains were compared to the corresponding chains of other representatives of the Carnivora order.     

Hemoglobins of reptiles. The primary structures of the alpha I- and beta I-chains of common iguana (Iguana iguana) hemoglobin

The primary structures of alpha I- and beta I-chains from the hemoglobins of the Common Iguana (Iguana iguana) are presented. The globin chains were separated on CM-cellulose in 8 M urea buffer. The amino-acid sequences were established by automatic Edman degradation of the native chains, the tryptic peptides and a peptide obtained by cyanogen bromide cleavage. The sequences are compared with human hemoglobin. Amino-acid replacements at positions critical for structure and function of the hemoglobin are discussed. The requirements for binding of ATP and also of DPG as allosteric effectors at the beta-chains seem to be fulfilled. Comparison of the alpha-chains with those of the Viper (Vipera aspis) shows 66 amino-acid substitutions. This number is in the same order of magnitude as the ones found by comparison with alpha-chains of crocodiles and mammals as well as with alpha A-chains of a turtle and birds. This result points towards a period of independent evolution of the reptile lines leading to the Common Iguana on one hand and to the Viper on the other. This time span is comparable to the one separating mammals from reptiles.     

Hemoglobins of reptiles. The primary structure of the major and minor hemoglobin component of adult Western Painted Turtle (Chrysemys picta bellii)

Red blood cells of adult Western Painted Turtles (Chrysemys picta bellii) contain two hemoglobin components: HbA (alpha A2 beta 2) and HbD (alpha D2 beta 2). We present the complete amino-acid sequences of the alpha A-chains from the major component and of the beta-chains common to both components. Structural features are discussed with respect to the animals extreme tolerance of severe hypoxic conditions during hibernation which is accompanied by a high oxygen affinity of the hemoglobin. The strong ATP dependence of Western Painted Turtle hemoglobin oxygen affinity is contrasted by the loss of one ATP-binding site, beta 143(H21)-Arg----Leu. The primary structure of the beta-chains excludes an allosteric control mechanism by hydrogencarbonate as it was found in crocodiles. Except in turtles a hemoglobin pattern with HbA and HbD sharing the same beta-subunits has been found only in birds. In comparison to other vertebrate hemoglobins there is a surprising similarity of the sequences to those of bird hemoglobins. alpha A- as well as alpha D-chains show larger homologies to chains of the same type in different species than alpha A- and alpha D-chains to each other in the same species. This indicates a duplication of the alpha-gene preceding the divergence of turtles and birds.     

Primary structure and functional properties of the hemoglobin from the free-tailed bat Tadarida brasiliensis (Chiroptera). Small effect of carbon dioxide on oxygen affinity

The hemoglobin of the Free-Tailed Bat Tadarida brasiliensis (Microchiroptera) comprises two components (Hb I and Hb II) in nearly equal amounts. Both hemoglobins have identical beta-chains, whereas the alpha-chains differ in having glycine (Hb I) or aspartic acid (Hb II) in position 115 (GH3). The components could be isolated by DEAE-Sephacel chromatography and separated into the globin chains by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by Edman degradation with the film technique or the gas phase method (the alpha I-chains with the latter method only), using the native chains and tryptic peptides, as well as the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond in the beta-chains. The comparison with human hemoglobin showed 18 substitutions in the alpha-chains and 24 in the beta-chains. 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 substituted. A comparison with other chiropteran hemoglobin sequences shows similar distances to Micro- and Megachiroptera. The oxygenation characteristics of the composite hemolysate and the two components, measured in relation to pH, Cl-, and 2,3-bis-phosphoglycerate, are described. The effect of carbon dioxide on oxygen affinity is considerably smaller than that observed in human hemoglobin, which might be an adaptation to life under hypercapnic conditions.