Inhibition of Bohr Effect after Removal of C-Terminal Histidines from Haemoglobin β-Chains

Kilmartin, John V.; Wootton, John F.

doi:10.1038/228766a0

Cited by 119 publications

(43 citation statements)

References 9 publications

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“…The "off" constant is almost independent of pH and, contrary to what is observed with HbA (14), there is no effect of IP6 on the "off" constant at either pH 7 or 9 ( The x-ray work of Perutz and coworkers (17) has suggested a structural picture of the role played by the C-terminal residues of the ( chains in determining the functional properties of hemoglobin. In particular, there is evidence for the involvement of the C-terminal histidine (( 146) in the Bohr effect (18) and of the penultimate tyrosine ((3 145) in the homotropic interaction shown by hemoglobin (17). The absence of hemeheme interaction of HbCPA in Bis-Tris may be correlated with the absence of the two penultimate tyrosines of the ( chains (17).…”

Section: Oxygen Kineticsmentioning

confidence: 87%

“…By stabilizing to a significant extent the lowaffinity conformation, otherwise essentially absent in HbCPA, IP6 thus provides the basis for the observed increase in n. To be sure, since evidence of the site or stoichiometry of 1P6 binding is lacking, no clear structural interpretation of this effect can be given. Nevertheless, apart from possible analogies with binding of organic phosphates to normal hemoglobin [ (17,18). Experiments on normal hemoglobin, however, suggest that IP6 may act indirectly to enhance the Bohr effect of HbCPA, just as it does in the case of unmodified hemoglobin (Fig.…”

Section: Oxygen Kineticsmentioning

confidence: 99%

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Partial Restoration of Normal Functional Properties in Carboxypeptidase A-Digested Hemoglobin

Bonaventura

Giardina

et al. 1972

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

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In the absence of organic phosphates human hemoglobin A digested with carboxypeptidase A (des His,.Tyr ,8) has high ligand affinity, a greatly reduced Bohr effect, and no heme-heme interaction. Under these conditions, it shows the simple, homogeneous ligandbinding kinetics characteristic of noncooperative heme proteins in which the high combination velocity for both 02 and CO accounts, to a large extent, for the increased affinity for both these ligands.Addition of inositol hexaphosphate dramatically alters the functional properties of this digested hemoglobin. The Bohr effect is greatly increased, and at neutral pH the protein shows significant, though still reduced, hemeheme interaction, together with a 5-fold decrease in affinity. In the presence of saturating amounts of the organic phosphate, the value of n is pH dependent, dropping from 1.9 at pH 5.8 to 1.3 at pH 8.6. After inositol hexaphosphate addition, the combination of the deoxy form of the digested hemoglobin with CO is 10-times slower than that observed in the absence of the inorganic phosphate; also the combination with CO after flash photolysis is biphasic and is similar, in many respects, to that observed for unmodified hemoglobin. Besides these functional changes, addition of inositol hexaphosphate to the modified deoxyhemoglobin results in an increase in the extinction coefficient at 430 nm similar to that observed on mixing the isolated a and # chains of normal hemoglobin. The results are consistent with the idea that inositol hexaphosphate shifts an equilibrium between high-and lowaffinity forms of the protein.Understanding regulatory phenomena in biological systems is one of the fundamental objectives of molecular biology. In the past few years it has become clear that control of enzymatic activity is often accomplished by means of conformational changes, and several allosteric models have been proposed to explain the role of such changes in determining the functional behavior of proteins. The results presented here are relevant to the problem of how conformational changes in hemoglobin, a prototype of an allosteric protein, can affect its functional properties.Digestion of human hemoglobin A by carboxypeptidase A removes the C-terminal residues of the # chains (histidine 146and tyrosine 145), with dramatic effects on the functional and structural properties of the molecule (1). The resulting hemoglobin, HbCPA, has a markedly reduced oxygen Bohr effect Abbreviations: HbCPA, hemoglobin A digested with carboxypeptidase A; IP6, inositolhexaphosphate; P2Glr, diphosphoglyceric acid; Bis-Tris, N,N-bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane. 2174and the heme-heme interactions are abolished (1). The large increase in ligand affinity, which makes HbCPA similar to the isolated chains, is independent of the nature of the ligand, the partition coefficient between oxygen and other ligands (e.g., CO) being similar to that of normal hemoglobin (2, 3). Digestion with carboxypeptidase A also produces large changes in the redox equilibrium, t...

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Section: Oxygen Kineticsmentioning

confidence: 87%

Section: Oxygen Kineticsmentioning

confidence: 99%

Partial Restoration of Normal Functional Properties in Carboxypeptidase A-Digested Hemoglobin

Bonaventura

Giardina

et al. 1972

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

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“…Hemoglobin stock solutions were stored as the carboxy derivative at 40, and used within 1 week of preparation. DesHis ,B146 hemoblogin was prepared by previously described procedures (2). The purified des-His ,B146 hemoglobin was fluorine-labeled by reaction with 1-trifluoro-3-bromoacetone, as described (5).…”

Section: Methodsmentioning

confidence: 99%

“…The importance of the carboxy terminal residues of the a and ( chains of hemoglobin to the Bohr effect and the cooperative mechanism has been suggested by studies of enzymatically modified hemoglobin (1,2) and hemoglobin mutants (3), and by features of the crystal structure (4). It has been proposed that electrostatic interactions involving the terminal side chains and carboxyls constrain the molecule into its deoxy form, and that the breaking of these bonds on initial ligand binding permits conformation transitions that expedite further ligand binding (4).…”

mentioning

confidence: 99%

Observation of Cooperative Ionizations in Hemoglobin

Huestis

Raftery

1972

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

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ABSTRACT'9F-Nuclear magnetic resonance studies of specifically fluorinated hemoglobin derivatives have been used to determine the apparent pKa of the histidine (3146 imidazole in deoxyhemoglobin. The titration of this residue was found to be abnormally sharp, particularly in the presence of diphosphoglyceric acid. The explanation advanced for this unusual titration curve may have implications for the mechanism of cooperative ligand binding. The possible role of such ionizations is discussed in light of some chemical evidence that the cooperative binding process is governed to a greater extent by internal nonpolar forces than by electrostatic interactions of exposed groups.The importance of the carboxy terminal residues of the a and ( chains of hemoglobin to the Bohr effect and the cooperative mechanism has been suggested by studies of enzymatically modified hemoglobin (1, 2) and hemoglobin mutants (3), and by features of the crystal structure (4). It has been proposed that electrostatic interactions involving the terminal side chains and carboxyls constrain the molecule into its deoxy form, and that the breaking of these bonds on initial ligand binding permits conformation transitions that expedite further ligand binding (4).We have labeled human hemoglobin specifically at cysteine (93 by reaction with 1-bromo-3-trifluoroacetone. The 19F-nuclear magnetic resonance (NMR) spectrum of this fluorinated residue reflects conformational processes in the region of the ali#2 interface where histidine (3146 (the carbon terminus) is bound to aspartate (394 in deoxyhemoglobin (HbdeO2). In an earlier communication (5), the preparation and functional properties of this trifluoroacetonylated hemoglobin (HbTFA) were described and changes in the 19F-NMR spectrum as a function of binding of ligands and allosteric effectors were discussed in detail. It was reported (5) HbTFAdeO2 had the appearance of a sharp titration curve, suggesting that the environment of the fluorine label was being influenced directly or indirectly by an ionizable group of pKa = 7.4. These earlier findings are summarized in Fig. 1.

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“…The digestion by carboxypeptidase A (CPA) was performed first under normal conditions [6], and, because of the very slow release of the involved residue, it was tried under more vigorous conditions, using an enzyme substrate ratio of 1: 1 as described by Perutz et al [7].…”

Section: Structural Studiesmentioning

confidence: 99%

Structural and functional study of Hb Nancy β 145 (HC 2) Tyr → Asp a high oxygen affinity hemoglobin

Gâcon¹,

Wajcman²,

Labie³

et al. 1975

FEBS Letters

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Inhibition of Bohr Effect after Removal of C-Terminal Histidines from Haemoglobin β-Chains

Cited by 119 publications

References 9 publications

Partial Restoration of Normal Functional Properties in Carboxypeptidase A-Digested Hemoglobin

Partial Restoration of Normal Functional Properties in Carboxypeptidase A-Digested Hemoglobin

Observation of Cooperative Ionizations in Hemoglobin

Structural and functional study of Hb Nancy β 145 (HC 2) Tyr → Asp a high oxygen affinity hemoglobin

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