An Estimation of the First Binding Constant of O2 to Human Hemoglobin A

Poyart, C; Bursaux, E; Bohn, B.

doi:10.1111/j.1432-1033.1978.tb12353.x

Cited by 28 publications

(21 citation statements)

References 38 publications

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“…Based on the extrapolation of the oxygen binding curves at very high and very low oxygen saturations, Shibayama and Saigo (2001) proposed the presence of a high oxygen‐affinity state with p50 of 1 to 2 torr and a low oxygen‐affinity state with p50 of 64 to 100 torr. These values are five‐ and twofold lower than those observed in solution for the binding of the first oxygen to T‐state hemoglobin in the absence and presence of allosteric effectors, respectively (Poyart et al 1978; Imai 1982).…”

mentioning

confidence: 69%

“…Deoxy‐hemoglobin was encapsulated in silica gels under conditions that, in solution, allow the attainment of either high or low oxygen affinities for T‐state hemoglobin, that is, in the absence and presence of the strong allosteric effectors bezafibrate and inositol hexaphosphate, respectively (Poyart et al 1978; Imai 1982). Oxygen binding curves were determined by measuring absorption spectra of hemoglobin silica gels as a function of oxygen pressure.…”

Section: Resultsmentioning

confidence: 99%

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High and low oxygen affinity conformations of T state hemoglobin

et al. 2001

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To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15°C, the oxygen pressure at half saturation (p50) was measured to be 12.4 ± 0.2 and 139 ± 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 ± 0.02 and 0.93 ± 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of ␣-and ␤-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high-and low-affinity T-state conformations.Hemoglobin is an ␣ 2 ␤ 2 -tetramer that binds ligands cooperatively via the modulation of equilibria between tertiary and quaternary conformations. X-ray crystallographic studies have shown that hemoglobin exists in two quaternary states, T and R, characterized by the presence and absence of salt bridges, respectively, and by a different pattern of ␣ 1 ␤ 2 -and ␣ 2 ␤ 1 -interactions (Perutz 1970;Baldwin and Chothia 1979). Binding of ligands triggers a series of tertiary conformational changes that break salt bridges, destabilizing the T state and leading to the liganded R state (Perutz 1970;Antonini and Brunori 1971;Perutz et al. 1998). Oxygen binding curves have been extremely useful in evaluating the effect of allosteric effectors on the functional properties of quaternary states (Imai 1982). In particular, the oxygen affinity and the degree of cooperativity of T-state hemoglobin have been investigated on mixed metal hybrids, for which iron at either the ␣-or ␤-heme was substituted with metals that do not bind oxygen (Shibayama

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confidence: 69%

Section: Resultsmentioning

confidence: 99%

High and low oxygen affinity conformations of T state hemoglobin

et al. 2001

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“…Supporting evidence for the identification of the Bohr groups has come from NMR (24,25), from hydrogen exchange experiments (26), from chemical reactivity studies (27,28), and from studies of mutant and modified hemoglobins (29)(30)(31)(32). Measurements indicate that the a1-a2 and intra-,B-chain Bohr groups contribute 60-80% of the total alkaline Bohr effect at normal (e.g., 0.1 M Cl-) salt concentrations (20,(29)(30)(31)(32)(33)(34). The remainder is thought to come from other chloride-linked sites (27,35,36).…”

Section: Formulation Of the Modelmentioning

confidence: 99%

“…Also, direct determinations of the pH dependence of the binding curve at very low and high oxygen pressures now exist (ref. 20; C. Poyart, personal communication).…”

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confidence: 99%

Structure-specific model of hemoglobin cooperativity.

Lee¹,

Karplus²

1983

Proc. Natl. Acad. Sci. U.S.A.

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A generalization of the Szabo-Karplus statistical mechanical model for hemoglobin cooperativity is formulated. The model fits the available thermodynamic and spectroscopic data with assumptions that are consistent with structural results and empirical energy function calculations. It provides a mechanism of hemoglobin cooperativity that is a generalization of the proposals of Monod, Wyman, and Changeux and of Perutz. The role of nonsalt-bridge related sources of constraints on ligand affinity and the mode of salt-bridge coupling to tertiary-quaternary structural changes are examined within the framework of the model. Analysis of proton release data for a range of pH values indicates that a pH-independent part of cooperativity must be present. The pH dependence of the first and last Adair constants point to partial linkage of salt bridges to ligation in the deoxy state and to a destabilized intra-13chain salt bridge in the unliganded oxy state.Hemoglobin has long been regarded as the prototype system for the investigation of cooperativity in macromolecules. The essential aim of such studies is to determine the detailed relationship between structural changes induced by ligation and the thermodynamic and kinetic manifestation of cooperativity. The x-ray structures of various hemoglobins solved by Perutz and his collaborators (1) have demonstrated that there exist two quaternary structures (deoxy and oxy) for the tetramer and two tertiary structures for each individual chain (liganded and unliganded). Based on the structural results and other data, Perutz (2, 3) proposed a stereochemical mechanism for cooperativity, in which salt bridges (some with ionizable protons in the neutral pH range) provide the link between ligand-induced tertiary structural changes and the relative stability of the two quaternary forms. To examine the thermodynamic correlates of the Perutz mechanism, its elements were incorporated into a statistical-mechanical model (4) (referred to as SK in what follows) that utilizes a partition function describing the influence of homotropic (oxygen) and heterotropic (protons and 2,3-bisphosphoglycerate) effectors on the set of contributing structures. It was found that the model was able to provide a satisfactory description of the cooperativity in ligand binding, the alkaline Bohr effect, the effect of 2,3-bisphosphoglycerate on ligand affinity, and the influence of chemical modifications and certain mutations on these properties (4-6). Further, the values of the model parameters, which correspond to physically meaningful quantities, were in the range suggested by independent estimates, thus providing support for the basic postulates of the Perutz mechanism.The structural, spectroscopic, and thermodynamic data on hemoglobin that have become available since the model was proposed call for its reevaluation at this time. Of particular importance are the structural results concerning the nature of tertiary-quaternary coupling. It is now clear from comparisons of crystal structures of unliganded d...

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“…Human hemoglobin A solution was prepared in the oxy form by usual procedures from whole blood as described previously [7,8]. Carbamoylated hemoglobin was obtained by the method described by Nigen et al [9].…”

Section: Methodsmentioning

confidence: 99%

Effect of Carbamination on the Buffering Power of Purified Human Hemoglobin A Solutions at Two Temperatures

1982

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The effect of COZ on hemoglobin buffering power was studied in purified human hemoglobin A solutions, native and specifically carbamoylated on N-terminal valines, at 26°C and 4 2 T , in the oxy and deoxy states. Titrations were performed by varying pco2 and by strong acid or base in the absence of C 0 2 . The participation of N-terminal valines to the total buffering power was estimated by subtracting the buffering value measured on carbainoylated hemoglobin solutions from that measured on native hemoglobin solutions.In the absence of COz the buffering value of native and modified hemoglobin increased slightly (Icss than lo:<) (a) on going from the deoxy to the oxy state, and (b) on raising the temperature from 26°C to 42°C.In the presence of COZ the buffering value of Hb increased from 9.1 to 16.6 mol mol HbT1 pH-' and that of HbOz from 10.1 to 19.6 mol mol H b i l pH-' when the temperature was raised from 26 ' C to 42 C. These figures correspond to a rise in the fraction of the total buffering value attributable to N-terminal valines from 1 1 7; to 25The present results point to a non-specific effect of COZ within the hemoglobin molecule independent of that of N-terminal valines. This effect nearly doubles the buffering value for CO2 when the temperature is raised, and contributes to pH regulation and COz removal in tissues with a high metabolic rate.for H b and from 3 'x to 33 yd for HbOz. Hemoglobin buffering power for non-volatile acids (Bny)is expressed differently from that for volatile acids (/jy), in this case C02. b,,, is defined as the slope of the titration curve of hemoglobin by strong base at a given pH, whereas /Iv is equal the negative slope of tlie hydrogen carbonate ion concentration versus pH curve of hemoglobin at a given pH. The buffering power of hernoglobin depends upon tlie presence of amino acids which ionize in the physiological pH range. These groups arc mainly tlie imidazole groups of the 22 titratable histidines of the polypeptide chains [I] and the a-amino groups of thc four N-terminal valines of the x and /i chains [2]. l n addition, oxygen (Or) and carbon dioxide ( C 0 2 ) binding to hemoglobin affccls the buffering value of the molecule. Indeed, in the physiological pH range, some specific groups (alkaline Bohr groups) undergo a lowering of their pk' upon hemoglobin oxygenation, which not only contributes to the alkaline Bohr effect but will also increase their buffcring power. These groups arc the two Val-lx, the two His-146P [ 3 ] and, to a still unsettlcd extent, other groups such as . The addition of C 0 2 to the solutions also influences hemoglobin buffcring power. In its presence, thc negative charge of the protein is increased and the pH is decreased owing to the binding of molecular CO2 to the x-amino groups of both valine-la and valinc-1B. This results in the formation of ionized carbamino compounds according to the following reactions [ S ] :HbNH:Ahhreviufions. Hb, deoxygenated hemoglobin A ; H b 0 2 , oxygenated hemoglobin A ; Hbc, dcoxygenated hemoglobin A reacted wit...

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An Estimation of the First Binding Constant of O2 to Human Hemoglobin A

Cited by 28 publications

References 38 publications

High and low oxygen affinity conformations of T state hemoglobin

High and low oxygen affinity conformations of T state hemoglobin

Structure-specific model of hemoglobin cooperativity.

Effect of Carbamination on the Buffering Power of Purified Human Hemoglobin A Solutions at Two Temperatures

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