Alteration of functional properties associated with the change in quaternary structure in unliganded haemoglobin

Kilmartin, John V.; Hewitt, J.A.; Wootton, John F.

doi:10.1016/0022-2836(75)90128-x

Cited by 110 publications

(76 citation statements)

References 35 publications

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“…Human adult Hb (Hb A), S-(N-ethylsuccinimido)cysteinyl (NES) des-Argl4la-Hb, and the isolated a and : chains were prepared as described (17). Cyanomet hybrid Hbs were prepared as reported (18).…”

Section: Methodsmentioning

confidence: 99%

Differences in Fe(II)-N epsilon(His-F8) stretching frequencies between deoxyhemoglobins in the two alternative quaternary structures.

Nagai¹,

Kitagawa²

1980

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

138

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Resonance Raman spectra have been obtained of the adeoxy and fldeoxy subunits within valency hybrid hemoglobins both in the high-affinity (R) and low-affinity (T) structures. Upon conversion from the R to the T structure, the vibrational frequency of the Fe(II-N(His-F8) bond changes from 223 to 207 or 203 cm-1 in the adeoxY subunit and from 224 to 220 or 217 cm-1 in the #deoxy subunit. We estimate that the Fe(II-N(His-F8) bond is stretched by the RT transition 3 times more in the a subunit (0.024 A) than in the ,B subunit (0.0085 A) and, accordingly, the strain energy developed in that bond is 8 times larger in the a than in the , subunit. Hence, the oxygen affinity of the a and # subunits may be regulated by different mechanisms.The oxygen affinity of Hb increases with the number of bound oxygen molecules. This increase in oxygen affinity, known as the heme-heme interaction, arises from a reversible transition between two alternative quarternary structures, the low-affinity (T) and high-affinity (R) structures (1, 2). The free energy of oxygen binding is larger by 3.6 kcal/mol (heme) in the R structure than in the T structure (3,4). Perutz (5) proposed that the equilibrium between the two quaternary structures depends on the distance between NE(His-F8) and the plane of the porphyrin. The T structure is more stable when the iron atom is penta-coordinated and when both the iron atom and the proximal His-F8 are displaced from the porphyrin plane. The stronger bonds between the subunits in the T structure might pull the proximal In some circumstances, the a and /3 subunits differ spectroscopically (7-14). In deoxyHb, the a subunit is mainly responsible for the changes in the Soret and visible absorption bands observed on R-oT transition (7,8). Perutz et al. (15,16) interpreted these spectral changes in terms of increased Fe(II)-N distance in the T structure. If the globin exerts a larger strain on the Fe(II)-NE(His-F8) bond in the a than in the / subunit, then the Fe(II)-NE(His-F8) stretching frequency should reveal it.We have measured resonance Raman spectra of various valency hybrid Hbs in the T and R structures. The excitation of Raman scattering at 441.6 nm enhanced only the Raman spectra of the ferrous subunit and thus allowed us to observe the R-T-linked structural changes of the heme in the adeoxy or deoxy subunit within valency hybrid Hbs. The R-T-linked frequency change of the Fe(II)-NE(His-F8) stretching Raman line was much larger in the a than in the /3 subunit. MATERIALS AND METHODSHuman adult Hb (Hb A), S-(N-ethylsuccinimido)cysteinyl (NES) des-Argl4la-Hb, and the isolated a and : chains were prepared as described (17). Cyanomet hybrid Hbs were prepared as reported (18). Hb M Milwaukee and Hb M Boston were purified by ion-exchange chromatography on an Amberlite IRC-50 (type II) column equilibrated with 0.1 M phosphate buffer (pH 7.0) (19). All Hb solutions were deionized by passage through a Dintzis column (20) and deoxygenated by repeated evacuation and flushing with N2 gas.Raman scattering...

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Section: Methodsmentioning

confidence: 99%

Differences in Fe(II)-N epsilon(His-F8) stretching frequencies between deoxyhemoglobins in the two alternative quaternary structures.

Nagai¹,

Kitagawa²

1980

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

138

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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%

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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“…HPLC analysis of a small aliquot of this reaction mixture demonstrated the removal of about 25% His-146␤. Des-His-146␤ chains were separated from undigested ␤ chains on a CM52 column as already described (18). Purity of the preparation was determined by analytical HPLC under conditions cited above and by polyacrylamide gel electrophoresis.…”

Section: Preparation Of Des-his-146␤ Hbamentioning

confidence: 99%

“…Preparation of des-His-146␤ HbA and removal of uncombined desHis-146␤ chains were accomplished as described previously (18). Purity of the des-His-146␤ HbA preparation was checked by polyacrylamide gel electrophoresis and analytical HPLC using a glass TSK gel DEAE-5PW anion exchange column under conditions described elsewhere (19).…”

Section: Preparation Of Des-his-146␤ Hbamentioning

confidence: 99%

Structure and Oxygen Affinity of Crystalline des-His-146β Human Hemoglobin in the T State

Bettati¹,

Kwiatkowski²,

Kavanaugh³

et al. 1997

Journal of Biological Chemistry

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To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146␤ human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146␤, the COOH-terminal histidine residues of the ␤ chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146␤ results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141␣ hemoglobin showed that removal of the COOH termini of the ␣ chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146␤ in the absence and presence of inositol hexaphosphate are 2.5-and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6-and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141␣ than those associated with His-146␤. It was demonstrated by Mozzarelli et al.(1) that crystals of human deoxyhemoglobin, grown from solutions of polyethylene glycol (PEG), 1 can bind oxygen reversibly. The T state hemoglobin molecules in these crystals have very low oxygen affinity that is comparable to the affinity measured for the binding of the first oxygen molecule to deoxyhemoglobin complexed with inositol hexaphosphate (IHP) in solution. Rivetti et al. (2) suggested that the low oxygen affinity of crystalline deoxyhemoglobin is due to crystal lattice stabilization of the T state structure so that the salt bridges associated with the Bohr effect are prevented from breaking upon ligand binding. This hypothesis is consistent with the absence of a Bohr effect in crystalline deoxyhemoglobin (2) and with the observation that the salt bridges associated with both the ␣-subunit and ␤-subunit COOH termini are intact in all of the partially liganded (3-7) and fully liganded (8, 9) T state x-ray crystal structures that have been determined to date.Evidence in support of the idea that the salt bridges associated with the COOH-terminal Arg-141␣ are a key determinant of the low oxygen affinity of crystalline T state hemoglobin comes from our previous studies (10 -12) on crystals of deoxyhemoglobin Rothschild (␤W37R) and des-Arg deoxyhemoglobin. Rivetti et al. (10) demonstrated that in the absence of chloride ions a 10-fold increase in oxygen affinity for crystalline deoxyhemoglobin Rothschild (␤W37R) is associated with a significant increase in the mobility of the ␣-subunit COOH termini in the deoxy crystal structure (11), but not with any detectable change in the stereochemistry of the d...

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Alteration of functional properties associated with the change in quaternary structure in unliganded haemoglobin

Cited by 110 publications

References 35 publications

Differences in Fe(II)-N epsilon(His-F8) stretching frequencies between deoxyhemoglobins in the two alternative quaternary structures.

Differences in Fe(II)-N epsilon(His-F8) stretching frequencies between deoxyhemoglobins in the two alternative quaternary structures.

Structure-specific model of hemoglobin cooperativity.

Structure and Oxygen Affinity of Crystalline des-His-146β Human Hemoglobin in the T State

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