Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Oxygen binding constants and stepwise enthalpies for human and bovine hemoglobin at pH 7.6.

A high-precision thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand-binding macromolecules in a manner analogous to that of the Gill thin-layer optical apparatus [Doleman & Gill (1976) Anal. Biochem. 87, 127]. We have generated differential heat-binding curves of oxygen binding to human and bovine hemoglobin in phosphate buffer at pH 7.6, with the enzyme-reducing system of Hayashi et al. [(1973) Biochim. Biophys. Acta 310, 309]. Experiments were conducted at a number of different temperatures in order to expand the data field, allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumptions of optical linearity between the various heme groups and reveals that the triply ligated species is measurably significant for both human and bovine hemoglobin. It was also determined that the concentration of doubly ligated species of bovine hemoglobin is relatively low. The experiments indicate that the reactions for both hemoglobins are enthalpy-driven for oxygen stepwise additions 1, 2, and 4 while being entropy-driven for step 3. Human hemoglobin oxygen-binding experiments were also performed with the Gill thin-layer optical apparatus under solution conditions identical to those used in the calorimeter. The experiments revealed that if optical linearity is assumed, the overall third equilibrium constant is negative or near zero. This indicated that either the optical cell's performance is much poorer than the thin-layer calorimeter or there is an appreciable nonlinear optical effect.     

Modulation of oxygen affinity in hemoglobin by solvent components. Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.     

Thin-layer microcalorimetric studies of oxygen and carbon monoxide binding to hemoglobin and myoglobin.

A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules. We have measured the enthalpy of binding oxygen and carbon monoxide to horse myoglobin, human hemoglobin A0 and sperm whale myoglobin in phosphate buffer at pH 7.6, with the enzyme reducing system of Hayashi. Reactions of human hemoglobin were also done under various buffer conditions in order to elucidate the Bohr effect. These binding reactions were found not to exhibit a detectable enthalpy change over the temperature range of 10 degrees C to 25 degrees C. The enzyme reducing system was shown to react with oxygen in a manner that releases a substantial amount of heat. This problem was corrected by using a minimum amount and by placing the buffer and enzyme system in the reference cell effectively cancelling the oxygen enzyme reaction heat as well as the heat of gas dissolution. It was also demonstrated that glucose-6-phosphate, one of the reducing system components, in 50 mM concentrations can influence the heat of binding oxygen and carbon monoxide to hemoglobin. This effect was shown to be absent in the myoglobins and also with hemoglobin at glucose-6-phosphate concentrations less than 5 mM.     

The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii)

• 1.1. Oxygen equilibrium curves were measured on trout red blood cell suspensions at pH 7.8 and 8.4 at 15, 20 and 25 C. Normal red cells and red cells that had been depleted of their ATP content were used.
• 2.2. The equilibrium data were fitted to the Adair's model and the enthalpy (ΔH) and entropy (ΔS) changes for the first and fourth steps of oxygenation and for overall oxygenation were calculated from the temperature dependencies of the Adair constants.
• 3.3. For normal red blood cells, the apparent heat for the first oxygenation step, δh₁, is close to zero.
• 4.4. Temperature insensitivity of this step at physiological pH, combined with a large pH dependence, probably denotes a property of Hb4, the Root effect Hb of trout blood.
• 5.5. At pH 7.8, ΔH₄ is about —4kcal/mol, a small value which may be attributed to the large release of Bohr protons that occurs at the last oxygenation step and corresponds to an endothermic process which opposes to the exothermic oxygenation of the haem.
• 6.6. The ΔH₄ value appears to have a large influence on the enthalpy for overall oxygenation.
• 7.7. Results for ATP-free red cells are consistent with a mere increase in the intracellular pH and suggest that ATP has no specific effect at and above pH_i ~ 7.7.
• 8.8. Effects of temperature and pH on trout red blood cell isotherms emphasize the primary importance of the major component of trout blood, namely Hb4, in trout blood functional properties.

     

Effects of trifluoperazine on calcium binding by calmodulin. A microcalorimetric study

Microcalorimetric titrations of calmodulin with Ca2+ and trifluoperazine (TFP) at various molar ratios have been carried out at 25 degrees C and at pH 7.0. Ca2+ binding to calmodulin produces heat (-delta H) in the presence of TFP, while heat is absorbed in the absence of TFP. The total heat produced by Ca2+ binding to all four sites is increased at increasing TFP-to-calmodulin ratios, attaining a plateau at about 7. These results indicate that at the higher ratios, the enthalpy changes (delta H) associated with Ca2+ binding are affected by TFP molecules bound at both high- and low-affinity sites. In addition, the Ca2+ binding reaction of the calmodulin-TFP complex is driven solely by a favorable enthalpy change of -27 kJ/mol of site; the entropy change (delta S) is -35 J/mol/K. These thermodynamic changes are opposite to those for TFP-free calmodulin and distinctly different from other Ca2+ binding proteins such as skeletal and cardiac troponin C and parvalbumin, where the reaction is driven by favorable changes of entropy as well as enthalpy.     

PH dependence of the Adair constants of human hemoglobin. Nonuniform contribution of successive oxygen bindings to the alkaline Bohr effect.

In order to solve the problem of an apparent discrepancy between the pH variance of oxygen equilibrium curve and the linear relation between the number of released Bohr protons and the degree of ligation, precise oxygen equilibrium curves of human hemoglobin were determined at a number of pH values from 6.5 to 8.8. From the equilibrium data individual steps (Adair constants), ki (i equals 1, 2, 3, 4), were obtained and the number of Bohr protons (deltaHi+) released on the ith stage of oxygenation was estimated. The pH dependence of k4 was very small, while the other ks strongly depended on pH over the pH range examined. As a consequence, the contribution of each step of oxygen binding to the alkaline Bohr effect nonuniform: deltaH4 was very small compared with deltaH1+, deltaH2+, and deltaH3+. In spite of this, calcuation has shown that the fractional number of released protons is essentially proportional to fractional oxygen saturation because of cooperative effects in hemoglobin. Thus, the present study indicates that the linear relationship between the fractional number of released protons and the degree of ligation, as obtained from titration experiments, is not necessarily incompatible with the pH variance of the shape of the oxygen equilibrium curve. The nonuniform pH depencence of the Adair constants implies that the two-state allosteric model of Monod, J., Wyman, J., and Changeus, J.P. (1965) J. Mol. Biol. 12, 88-118 is not adequate to describe the heterotropic effect caused by protons.     

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin.

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygenation properties of hemoglobin from the earthworm, Lumbricus terrestris. Effects of pH, salts, and temperature

Oxygen equilibrium curves of the extracellular hemoglobin from Lumbricus terrestris were determined under a variety of conditions. These data were characterized by (i) a rather small free energy of cooperativity (1.6-2.8 kcal/mol), (ii) a large and strongly pH-dependent Hill coefficient with a maximum value of 7.9, (iii) a high sensitivity of the upper asymptote of the Hill plot to pH, and (iv) a maximum association constant as large as that of the free beta subunit of human hemoglobin A. The effects of LiCl, KCl, NaCl, BaCl2, CaCl2, SrCl2, and MgCl2 on the oxygen equilibrium were measured. Cations, not Cl-, were found to control oxygen binding. Divalent cations have a larger effect on oxygen affinity than monovalent cations, and their effectiveness decreased in the order listed above within each valence class. These specific effects depend in part on ionic radius and cannot be explained in terms of ionic strength. The data indicate that the oxygenation-linked binding of a Ca2+ ion is accompanied by the release of two protons; the binding of a Na+ ion is associated with the release of one proton. These findings indicate that the oxygenation-linked cation-binding site contains two acid groups that do not readily dissociate their protons except when replaced by cations. Incubation at either pH 6.2 or 8.9 had no effect on subsequent measurements of oxygen equilibria at pH 7.8. The apparent heat of oxygenation was found to be -11.8, -7.3, and -9.3 kcal/mol at pH 9.0, 7.4, and 6.6, respectively. These differences indicate that proton-binding processes contribute to the heat of oxygenation.     

Effect of temperature on functional properties of carp hemoglobin

Precise oxygen equilibrium curves of carp hemoglobin have been obtained in 0·1 m-phosphate from 10 to 25 °C. The equilibrium data were analyzed according to the stepwise oxygenation model of Adair (1925) to obtain the enthalpy change (ΔH_i), entropy change (ΔS_i) and free energy change (ΔG_i) for the i (= 1, 2, 3, 4) individual oxygenation steps. The values of ΔH_i are definitely non-uniform with dependencies on i and the pH of the medium. The co-operative effects in carp hemoglobin are due mainly to enthalpic contributions under the conditions studied here. The thermodynamic properties suggest a structural transition with pK ~8·5 as was also seen in other functional and spectroscopic measurements.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors.

The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to estimate the enthalpy and entropy parameters for the formation of the corresponding 1:1 adducts. The obtained results indicate that binding of these anions to the cobalt enzyme is an enthalpy driven process which is opposed by a moderate entropy change.     

Calorimetric measurement of stepwise enthalpy changes for the binding of four oxygens to adult human hemoglobin

The successive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin have been measured by direct calorimetry at pH 7.4 and 6°. Average results in kcal/(mole O₂) are: ΔH₁ = ?25.1 ± 2.8; ΔH₂ = ?12.6 ± 3.0, ΔH₃ = ?12.5 ± 3.0, and ΔH₄ = ?10.1 ± 1.4. These results imply a substantial temperature dependence for the cooperativity of O₂ binding by the protein and generally resemble the van't Hoff results by Roughton $\text{et al}$. [Roy. Soc. of London Proc., $\text{B 144}$, 29 (1955)] for sheep hemoglobin at pH 9.1 and a temperature range of 2° to 19°.     

Direct observation of the enthalpy change accompanying the native to molten-globule transition of cytochrome c by using isothermal acid-titration calorimetry

The enthalpy change accompanying the reversible acid-induced transition from the native (N) to the molten-globule (MG) state of bovine cytochrome c was directly evaluated by isothermal acid-titration calorimetry (IATC), a new method for evaluating the pH dependence of protein enthalpy. The enthalpy change was 30 kJ/mol at 30 degrees C, pH 3.54, with 500 mM KCl. The results of the global analysis of the temperature dependence of the excess enthalpy from 20 to 35 degrees C demonstrated that the N to MG transition is a two-state transition with a small heat capacity change of 1.1 kJ K(-1) mol(-1). The present findings were also indicative of the pH dependence of the enthalpy and the heat capacity of the MG state, -13 kJ mol(-1) pH(-1) and -1.0 kJ K(-1) mol(-1) pH(-1), respectively, at 30 degrees C within a pH range from 2 to 3.     

Thermodynamic aspects of the co-operativity in four-step oxygenation equilibria of haemoglobin

Accurate oxygen equilibrium curves of human haemoglobin (concentration, 600 μm as haem) were determined by an automatic recording method (Imai et al., 1970) under a variety of conditions combining six different temperatures with seven sets of solute conditions, producing wide-ranging structural constraints on haemoglobin. The heat and entropy change of oxygenation for four individual steps (ΔH_tand ΔS_i, i = 1 to 4) were evaluated by a least-squares method directly from each set of six equilibrium curves without knowing the values of the four equilibrium constants k_t. As shown in previous studies with dilute haemoglobin solutions (Imai &; Tyuma, 1973; Imai &; Yonetani, 1975b) ΔH_i depended strongly on i; small amounts of heat were liberated at oxygenation steps involving the release of H⁺ and anions such as Cl^?, 2,3-diphosphoglycerate, and inositol hexaphosphate, while large amounts of heat were liberated on the oxygenation of the R state or highly constrained T state, from which no or few non-haem ligands are released. The observed amounts of heat, when corrected for the heat of H⁺ and anion release associated with oxygenation, became uniform to a good approximation, indicating that the intrinsic heat of haem oxygenation is essentially equal for the four oxygenation steps, and a large part of the non-uniformity of ΔH_i may be ascribed to the oxygen-linked release of the non-haem ligands. ΔS_i exhibited similar behaviour. The relation, k₁ ? k₂ ? k₃ ? k₄ which usually holds under physiological conditions, is a consequence of the presence of an enthalpy-entropy compensation process at the first three steps and its absence at the fourth step. The compensation temperature was around 300 K. The origin of the co-operativity cannot be specified as either an enthalpic or entropic effect. In the presence of 0.1 m-Cl^? and 2 mm-2,3-diphosphoglycerate, the T to R transition at any oxygenation step is an endothermic process and haemoglobin gains entropy on the transition. The deoxy T structure is stabilised by the enthalpy term, while the oxy R structure is stabilised by the entropy term, so that the T to R transition occurs at a stop where the entropy contribution exceeds the enthalpy contribution. The present study shows that the oxygen-linked binding of non-haem ligands is very important in co-operative oxygen binding by haemoglobin, as predicted by Perutz, (1970).     

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., and Robert, C. H. (1987) Biochemistry, 26, 3995-4002), the heats for binding two and four oxygen molecules were found to be simple multiples of the first binding heat. A direct consequence of equal stepwise heats is invariance of the shape of the binding curve with temperature, as pointed out by Wyman (Wyman, J. (1939) J. Biol. Chem. 127, 581-599). Titration microcalorimetry was also performed for the binding of carbon monoxide to hemoglobin. While the tight binding of CO precludes high-precision binding measurements, it does allow one to accurately determine the heat of ligation as a function of the CO bound. In these titrations a uniform heat of reaction is not observed, but the heat of binding increases markedly near the end point. This implies that the stepwise binding enthalpy for adding the third CO molecule is anomalously endothermic and for adding the fourth strongly exothermic. A similar phenomenon cannot be ruled out in the case of oxygen because of imprecision intrinsic in the analysis of the weaker ligand binding.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...     

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.