Discontinuous release of heat at successive steps of oxygenation in human and bovine hemoglobin at pH 9.0

We have measured the temperature dependence of the oxygen-binding isotherms of human and bovine hemoglobin at pH 9.0 in 0.1 M borate buffer. In both hemoglobins the ionization of the Bohr protons is finished at this pH; therefore, their heat does not interfere with the measurements. Two sets of curves have been obtained, which have been analyzed by either singular or global procedures for estimating the enthalpy changes of subsequent steps of oxygenation. The data indicate that in human hemoglobin the reaction with oxygen is enthalpy driven for steps 1, 2, and 4 while it is entropy driven for step 3. In bovine hemoglobin this phenomenon is even more evident: steps 2 and 4 are enthalpy driven while steps 1 and 3 are entropy driven. The discontinuous distribution of heat at subsequent steps of oxygenation suggests that the T to R transition in hemoglobin is not a monotonic process and involves conformations with novel characteristics.     

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

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

Allosteric effect of water in fish and human hemoglobins

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

The pH dependence of the binding of D-glycerate 2,3-bisphosphate to deoxyhemoglobin and oxyhemoglobin. Determination of the number of binding sites in oxyhemoglobin.

The number of Bohr protons released upon oxygenation has been measured over a large range of human hemoglobin concentrations (0.02 to 4.5 mM) in the presence of equimolar amounts of D-glycerate 2,3-bisphosphate. From these data the association constants for the binding of this organic phosphate to deoxyhemoglobin and oxyhemoglobin were calculated at different pH values. The maximum number of protons absorbed upon binding to oxyhemoglobin was determined as well. The maximum number of protons bound to deoxyhemoglobin upon binding of D-glycerate 2,3-bisphosphate was measured independently. From the pH dependence of the association constants and the maximum number of protons absorbed it could be concluded that only one D-glycerate 2,3-bisphosphate can be bound to both deoxyhemoglobin and oxyhemoglobin.     

Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Temperature dependence of oxygen binding and pH dependence of subunit dissociation.

The temperature dependence of the oxygen equilibrium of tadpole hemoglobin has been determined between 0 degrees and 32 degrees for the unfractionated but phosphate-free lysate and between 12 degrees and 32 degrees for each of the four isolated components between pH 6 and 10 in 0.05 M cacodylate, Tris, or glycine buffers containing 0.1 M NaCl and 1 mM EDTA. Under these conditions the Bohr effect (defined as deltalog p50/deltapH) of the unfractionated lysate is positive at low temperatures between pH 6 and 8.5 and is negative above pH 8.5 to 8.8 at any temperature. As the temperature rises the Bohr effect below pH 8.5 changes greatly. In the interval pH 7.0 to 7.5, the magnitude of the Bohr effect decreases from + 0.28 at 0 degrees to zero at about 24 degrees and becomes negative, as in mammalian hemoglobins, above this temperature. Measurements with the isolated components show that the temperature dependence of oxygen binding for Components I and II and for Components III and IV is very similar. For both sets of components the apparent overall enthalpy of oxygenation at pH 7.5 is about -16.4 kcal/mol and -12.6 kcal/mol at pH 9.5. The measured enthalpies include contributions from the active Bohr groups, the buffer ions themselves, the hemoglobin groups contributing buffering, and any pH-dependent, oxygenation-dependent binding of ions such as chloride by the hemoglobin. The apportioning of the total enthalpy among these various processes remains to be determined. Between pH 8 and 10.5 tadpole oxyhemoglobin undergoes a pH-dependent dissociation from tetramer to dimer. The pH dependence of the apparent tetramer-dimer dissociation constant indicates that at pH 9.5 the dissociation of each tetramer is accompanied by the release of approximately 2 protons. In this pH range the oxygen equilibrium measurements indicate that about 0.5 proton is released for each oxygen molecule bound. The results are consistent with the conclusion that one acid group per alphabeta dimer changes its pK from about 10 to 8 or below upon dissociation of the tetramer.     

Binding of acellular, native and cross-linked human hemoglobins to haptoglobin: enhanced distribution and clearance in the rat

It is well established that hemoglobin resulting from red cell lysis binds to haptoglobin in plasma to form a complex. The increased molecular size precludes its filtration by the kidneys, redirecting it toward hepatocellular entry. Chemically cross-linked hemoglobins are designed to be resistant to renal excretion, even in the absence of haptoglobin. The manner in which binding to haptoglobin influences the pharmacokinetics of acellular cross-linked and native hemoglobins was investigated after intravenous injection of radiolabeled native human hemoglobin and trimesyl-(Lys82)beta-(Lys82)beta cross-linked human hemoglobin, at trace doses, into rats. Under these conditions, there is sufficient plasma haptoglobin for binding with hemoglobin. In vitro binding assayed by size-exclusion chromatography for bound and free hemoglobin revealed that, at <8 muM hemoglobin, native human hemoglobin was completely bound to rat haptoglobin, whereas only approximately 30% of trimesyl-(Lys82)beta-(Lys82)beta cross-linked hemoglobin was bound. Plasma disappearance of low doses (0.31 mumol/kg) of native and cross-linked hemoglobins was monoexponential (half-life = 23 and 33 min, respectively). The volume of distribution (40 vs. 19 ml/kg) and plasma clearance (1.22 vs. 0.4 ml.min(-1).kg(-1)) were higher for native than for cross-linked hemoglobin. Native and cross-linked human hemoglobins were found primarily in the liver, and not in the kidney, heart, lung, or spleen, mostly as degradation products. These pharmacokinetic findings suggest that the binding of hemoglobin to haptoglobin enhances its hepatocellular entry, clearance, and distribution.     

The structure and function of plant hemoglobins.

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.     

Sulfhydryl groups and the structure of hemoglobin

1. Addition of 2 moles of mersalyl, mercuric chloride, p-chloromercuribenzoate (PCMB), or methyl mercury hydroxide per mole of hemoglobin greatly reduces heme-heme interactions (n), yet these substances have quite different effects on the oxygen affinity (-log p₅₀). Mersalyl and mercuric chloride at this concentration each increase the oxygen affinity, while PCMB and methyl mercury have little or no effect on the oxygen affinity. These effects are primarily associated with the binding of —SH groups, and are largely reversed on the addition of glutathione. —SH groups do not appear to be responsible for the Bohr effect. 2. Evidence is presented for the belief that the two hemes of each half-molecule of horse hemoglobin are situated on either side of a cluster of—SH groups. 3. The mechanism of interaction between the hemes is discussed. It is concluded that the reorganization of the protein architecture which accompanies oxygenation plays a central role in this interaction, in agreement with the views of Pauling and Wyman.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Immunological relatedness of annelid extracellular hemoglobins and chlorocruorins

1. The immunological relatedness of several annelid extracellular hemoglobins and chlorocruorins was investigated using ELISAs and Western blotting to determine the binding of purine polyclonal and monoclonal antibodies to Lumbricus terrestris hemoglobin with the hemoglobins of Tubifex tubifex, Tylorrhynchus heterochaetus, Arenicola marina and Macrobdella decora and the chlorocruoins of Myxicola infundibulum and Eudistylia vancouverii. 2. Polyclonal antibodies to Lumbricus terrestris hemoglobin bound to all the other hemoglobins and chlorocruorins. However, the titers were in all cases one to several orders of magnitude smaller than with Lumbricus terrestris hemoglobin. 3. Polyclonal antibodies to Eudistylia vancouverii chlorocruorin bound to the hemoglobins of Lumbricus terrestris, Tubifex tubifex, Arenicola marina, Tylorrhynchus heterochaetus and Macrobdella decora. 4. Of the nine monoclonal antibodies to Lumbricus terrestris hemoglobin isolated, two (No. 24 and No. 26) bound to the other hemoglobins and to Myxicola chlorocruorin, but the binding was again weaker than with Lumbricus hemoglobin. Antibody No. 26 also bound to Eudistylia chlorocruorin. Although antibody No. 24 appears to recognize a conformation-dependent epitope, antibody No. 26 recognizes a common epitope in each of the four subunits M, D1, D2, and T of unreduced Lumbricus hemoglobin. 4. An additional two monoclonal antibodies to Lumbricus hemoglobin (No. 21 and No. 25) bound also only to Tubifex hemoglobin. Antibody No. 21 recognizes subunits D1 and M of Lumbricus hemoglobin and No. 25 appears to recognize a conformation-dependent epitope.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

Preparation and characterization of dimeric bovine hemoglobin tetramers

Dimeric bovine hemoglobin (Hb) tetramers were prepared by a one-step solid phase adsorption method. Briefly, Hb was absorbed by the solid phase, Q Sepharose Fast Flow media, followed by reaction with the glutaraldehyde and elution procedure. Then, dimeric bovine Hb tetramers were formed and purified from Hb tetramers by anion-exchange chromatography based on Protein-Pak DEAE 8HR. The dimeric Hb tetramer showed a P50 value of 15.9 mm Hg, oxygen transporting efficiency of 14.2%, and Hill coefficient of 1.72. The number of Bohr protons released for dimeric Hb tetramers was 0.59 H/tetramer, which was 39% of that of native bovine Hb. The number of chloride ions released on oxygenation was 0.60/tetramer for dimeric Hb tetramers, which was 46% of that of native bovine Hb.     

Comparative immunochemical studies of primate hemoglobins

The antigenic properties of a number of chromatographically purified primate hemoglobins were compared to those of normal human hemoglobin using a sensitive radioimmunochemical procedure. The degree of inhibition of the antigen-antibody reaction with heterologous hemoglobins appeared to be related to the structural similarity of these proteins to the normal human hemoglobin immunogen. With the exception of the baboon hemoglobin, the antigenicity of the hemoglobins paralleled the phylogeny of the primates. The gorilla and chimpanzee hemoglobins were antigenically identical to normal human hemoglobin, whereas the gibbon and orangutan hemoglobins were substantially more variable. Of the Old World monkey hemoglobins examined, the baboon produced lower inhibition values, suggesting a greater degree of structural dissimilarity than other Cercopithecoidea hemoglobins, which is compatible with a greater rate of evolutionary change occurring in this protein. Using the known amino acid sequences of human and other primate hemoglobins, we have attempted to identify antigenic determinant areas of the proteins.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : III. Immunochemical Detection of Tadpole and Frog Hemoglobins (Rana catesbeiana) in Single Erythrocytes

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.     

Separation of asymmetrical hybrid hemoglobins by anaerobic cation-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of two structurally different hemoglobins were found to be readily separated by cation-exchange high-performance liquid chromatography under anaerobic conditions. When oxyhemoglobins A and S were mixed and deoxygenated, the resulting HPLC chromatogram showed three peaks. The distribution of the three components follow the binomial expansion a² + 2 ab + b² = 1, where a and b are the initial fractions of parent hemoglobins. The middle peak was collected in a test tube saturated with CO gas and reanalyzed under the same experimental conditions. This middle component gave two peaks of equal areas with retention times identical to those of the CO-form of the parent hemoglobins without the appearance of the hybrid hemoglobin band. No intermediate peak was observed in solutions of mixtures of liganded hemoglobins under aerobic conditions. Hybrid hemoglobins AC and SC were also formed when oxyhemoglobins A and C, S and C were mixed, respectively. The separation and the identification of hemoglobins and hybrid hemoglobin employing cation-exchange HPLC can be achieved within 30 min by gradient elution. In addition, the ability to isolate hybrid hemoglobins may be a valuable tool for the study of physical and chemical properties of hybrid hemoglobins.     

A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1.

Lepore hemoglobins result from crossovers between normal beta and delta chain genes. Structural investigation of two newly discovered examples of Lepore hemoglobins revealed one of them to be structurally identical to hemoglobin Lepore Hollandia α₂^Aδ²² -x- β⁵⁰, a rarely occurring Lepore variant, while the second had the structure of hemoglobin Lepore Boston α₂^Aδ⁸⁷ -x- β¹¹⁶. Studies of the equilibrium and kinetic properties of the liganding reactions of these two Lepore hemoglobins, which differ only in three amino acid residues, and comparison of these with the known properties of hemoglobin A₁ (α₂β₂) and hemoglobin A₂ (α₂δ₂) have been carried out. A high value of n, the Hill coefficient, indicating normal heme-heme interaction, was observed in each hemoglobin along with a normal Bohr effect. However, a slight but definite increase in oxygen affinity was observed for each Lepore hemoglobin. Furthermore, kinetic studies indicated a slight but consistently increased rate of ligand combination and a somewhat decreased rate of oxygen dissociation for hemoglobins Lepore Hollandia and Lepore Boston at pH 7 and 20 °C. Apparently, the higher oxygen affinity of these Lepore hemoglobins over those of the normal hemoglobins A₁ and A₂ reflects changes of sequence that are common to both types of hemoglobin Lepore.     

Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes.

The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.     

A model of the pH-dependence of the number of oxygen-linked chloride binding sites in hemoglobin.

A model is presented of the pH-dependence of the number of oxygen-linked chloride binding sites established by nuclear magnetic resonance quadrupole-relaxation studies on various mutant and chemically modified hemoglobins. The predictions of the model are in good qualitative agreement with the measured pH-dependences of the linewidth of the 35Cl- NMR signal. The obtained agreement implies that more chloride is bound to oxygenated than to deoxygenated hemoglobin.     

Interaction of triethyltin with cat hemoglobin: Identification of binding sites and effects on hemoglobin function

Binding of triethyltin to the cat hemoglobins (HbA and HbB) results in the “masking” of two of the freely reactive sulfhydryl groups (SH) within the hemoglobin tetramer. That the “masked” SH groups occur in position 13α of each α-subunit was demonstrated by the lack of labeling of cysteine 13α with [¹⁴C]N-ethylmaleimide when triethyltin is present. Studies with cat-human hybrid hemoglobins indicate that the α-subunit of the cat hemoglobins alone is involved in the formation of a complex with triethyltin. Using available data on the primary as well as three dimensional structures of animal hemoglobins, it is suggested the cysteine 13α and histidine 20α serve as axial ligands in the formation of a pentacoordinate triethyltin cat hemoglobin complex. The binding of triethyltin results in an increase in the oxygen affinity of the two cat hemoglobins.     

The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate.

The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar (Lepisosteus osculatus), and of four teleost fish, the carpsucker (Carpiodes carpio), the small mouth buffalo fish (Ictiobus bubalus), the Rio Grande cichlid (Cichlasoma cyanoguttatum), and the redear sunfish (Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μm ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO₂ for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect ($\text{Δlog}\text{P}{}_{30}\text{ΔpH}$) in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μm ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in $\text{Δlog}\text{P}{}_{50}\text{ΔpH}\text{~ 0.3}$). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation.