T-state hemoglobin with four ligands bound

Flash photolysis kinetics have been measured for ligand recombination to hemoglobin (Hb) in the presence of two effectors: bezafibrate (Bzf) and inositol hexakisphosphate (IHP). The combined influence of the two independent effectors leads to predominantly T-state behavior. Samples equilibrated with 0.1 atm of CO are fully saturated, yet after photodissociation they show only T-state bimolecular recombination rates at all photolysis levels; this indicates that the allosteric transition from R to T occurs before CO rebinding and that the allosteric equilibrium favors the T-state tetramer with up to three ligands bound. Since all four ligands bind at the rate characteristic for the T-state, the return transition from T to R must occur after the fourth ligand was bound. At 1 atm of CO, rebinding to the initial R state competes with the allosteric transition resulting in a certain fraction of CO bound at the rate characteristic for the R state; this fraction is greater the smaller the percentage dissociation. Under 1 atm of oxygen, samples are not more than 93% saturated and show mainly T-state kinetics. The results show that all four hemes can bind oxygen or CO ligands in the T structure. The fraction of the kinetics occurring as geminate is less for partially liganded (T-state) samples than for fully liganded (R-state) Hb.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.     

Flash photolytic studies of carbon monoxide binding to the ferrous chains of [Mn(II),Fe(II)] hybrid hemoglobins: kinetic mechanism for the early stages of hemoglobin ligation

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the alpha chains or beta chains. At elevated pH, CO binding to the alpha chains produces a larger degree of T to R conversion than binding to the beta chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both alpha- and beta-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.     

Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the alpha chains (alpha alpha Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of alpha alpha Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of alpha alpha-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state alpha alpha Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for alpha alpha Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state alpha alpha Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for alpha alpha Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.     

Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery

The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.     

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.     

Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids.The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.     

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.     

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.     

Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids.

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Control of the allosteric equilibrium of hemoglobin by cross-linking agents

The kinetics of ligand rebinding have been studied for modified or cross-linked hemoglobins (Hbs). Several compounds were tested that interact with alpha Val 1 or involve a cross-link between alpha Val 1 and alpha Lys 99 of the opposite dimer. By varying the length of certain cross-linking molecules, a wide range in the allosteric equilibrium could be obtained. Several of the mono-aldehyde modified Hbs show a shift toward the high affinity conformation of Hb. At the other extreme, for certain di-aldehyde cross-linked Hbs, the CO kinetics are typical of binding to deoxy Hb, even at low photodissociation levels, with which the dominant photoproduct is the triply liganded species; in these cases the hemoglobin does not switch from the low to high affinity state until after the fourth ligand is bound. Although each modified Hb shows only two distinct rates, the kinetic data as a function of dissociation level cannot be simulated with a simple two-state model. A critical length is observed for the maximum shift toward the low affinity T-state. Longer or shorter lengths of the cross-linker yielded more high affinity R-state. Unlike native Hb, which is in equilibrium with free dimers, the cross-linked Hbs maintain the fraction slow kinetics, which is unique to Hb tetramers, even at 0.5 microM (total heme). Addition of HbCN to unmodified HbCO solutions results in dimer exchange, which decreases the relative fraction of slow bimolecular kinetics; the cross-linked Hbs did not show such an effect, indicating that they do not participate in dimer exchange.     

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.     

The primary structure of the hemoglobin of the European Souslik (Citellus citellus, Rodentia)

The complete primary structures of two hemoglobin components of the European Souslik (Citellus citellus) are presented. The two hemoglobins have identical alpha-chains but differ in the amino-acid sequence of their beta-chains. The chain separation was achieved by chromatography on carboxymethyl-cellulose CM-52. Amino-acid sequences were established by automatic liquid-phase and gas-phase Edman degradation of the globin chains, of their tryptic peptides and of a peptide resulting from acidic hydrolysis of an Asp-Pro bond in the alpha-chain. The differences between the two beta-chains are manifested in three amino-acid exchanges. The sequences are compared with those of human and European Marmot hemoglobins. Only few differences were found among hemoglobins of C. citellus and other representatives of Sciuromorpha.     

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 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.     

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.     

The primary structure of hemoglobin from goldfish (Carassius auratus)

The primary structures of the alpha- and beta-chains from goldfish hemoglobin are given. The globin chains were separated by gel filtration after air-oxidation of globin. After chemical and enzymatical cleavage of the chains, the peptides were isolated by gel filtration and ion exchange chromatography on Dowex. The fish-chains have one residue more than the human chains. The alpha-chain is acetylated at the amino-terminal residue and has no cysteine. Compared with the human chains there are 66 amino-acid differences in the alpha- and 72 in the beta-chains. The implication of these differences for the physiology of the hemoglobin molecule of goldfish is discussed.     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

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.