Tetramer‐dimer equilibrium of oxyhemoglobin mutants determined from auto‐oxidation rates

Griffon, Nathalie; Baudbin, Véronique; Dieryck, Wilfrid; Dumoulin, Antoine; Pagnier, J.; Poyart, Claire; Marden, Michael C.

doi:10.1002/pro.5560070316

Cited by 53 publications

(45 citation statements)

References 64 publications

(84 reference statements)

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“…7D). Interestingly IHP, an allosteric effector that also pushes the tetramer-dimer equilibrium toward tetramer at low concentrations, but toward dimers when used at high concentrations as was the case in our studies (42)(43)(44)(45)(46)(47)49), showed an increased observed rate constant similar to those seen by dimers at both 50 and 25 M ProliNO (see Fig. 5).…”

Section: Effects Of T-state Stabilization and Ph Onsupporting

confidence: 54%

“…Experiments run at 15°C confirmed this trend, with IHP being more effective at increasing the rate constants at lower temperature. The reasons for these temperature effects of IHP on rates are not clear, but we speculate that IHP has two effects: it speeds up the reaction through the stabilization of the T-state but also may promote the formation of HbA dimers when used at high concentrations (42)(43)(44)(45)(46)(47). These hemoglobin dimers can also cause the reaction to speed up as discussed below.…”

Section: Effects Of T-state Stabilization and Ph Onmentioning

confidence: 99%

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Low NO Concentration Dependence of Reductive Nitrosylation Reaction of Hemoglobin

Tejero

Basu

Helms

et al. 2012

Journal of Biological Chemistry

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Background:Interactions of nitric oxide with hemoglobin are critical to NO function. Results: Reductive nitrosylation of hemoglobin is faster at low NO concentrations and sensitive to allosteric modifications. Conclusion: Hemoglobin can catalyze faster reductive nitrosylation reactions at physiological, low NO concentrations. Significance: Reductive nitrosylation may be more relevant in vivo than previously recognized.

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Section: Effects Of T-state Stabilization and Ph Onsupporting

confidence: 54%

Section: Effects Of T-state Stabilization and Ph Onmentioning

confidence: 99%

Low NO Concentration Dependence of Reductive Nitrosylation Reaction of Hemoglobin

Tejero

Basu

Helms

et al. 2012

Journal of Biological Chemistry

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“…Higher order multimers are not necessarily observed using other methods such as SDS-PAGE [47]. The tetramers and dimers cannot be separated by chromatography because they interchange on a time scale of seconds [48]. However, careful fitting of chromatographic peak shapes allows detection of the dimers and determination of the dissociation equilibrium constant.…”

Section: Mass Spectra Of Hemoglobinmentioning

confidence: 99%

Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

Wright

Douglas

2009

J. Am. Soc. Mass Spectrom.

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The conformations of gas-phase ions of hemoglobin, and its dimer and monomer subunits have been studied with H/D exchange and cross section measurements. During the H/D exchange measurements, tetramers undergo slow dissociation to dimers, and dimers to monomers, but this did not prevent drawing conclusions about the relative exchange levels of monomers, dimers, and tetramers. Assembly of the monomers into tetramers, hexamers, and octamers causes the monomers to exchange a greater fraction of their hydrogens. Dimer ions, however, exchange a lower fraction of their hydrogens than monomers or tetramers. Solvation of tetramers affects the exchange kinetics. Solvation molecules do not appear to exchange, and solvation lowers the overall exchange level of the tetramers. Cross section measurements show that monomer ions in low charge states, and tetramer ions have compact structures, comparable in size to the native conformations in solution. Dimers have remarkably compact structures, considerably smaller than the native conformation in solution and smaller than might be expected from the monomer or tetramer cross sections. This is consistent with the relatively low level of exchange of the dimers. . Electrospray ionization has allowed the study of protein ions in the gas phase, free of water. In the absence of water, monomeric proteins can adopt compact conformations similar to the solution conformations, but also extended conformations far removed from the native state [2,3]. Much of the current understanding of the "size" of protein ions in the gas phase comes from physical methods of measuring collision cross sections using ion mobility spectrometry at pressures of a few Torr [4 -7], at atmospheric pressure [8], or at pressures of ca. 1 ϫ 10 Ϫ3 Torr [7,9,10]. Measurements of ion kinetic energy loss in triple quadrupole systems have also been used to determine collision cross sections of proteins [11][12][13] and protein-ligand complexes [11,14,15]. Chemical probes of protein conformation have been used as well, with much of the work using gas-phase hydrogen deuterium exchange (H/Dx) of trapped ions, either in linear [16 -19] or 3D [20] quadrupole ion traps, or ion cyclotron resonance (ICR) mass spectrometers [21][22][23][24][25].The study of the structure of monomeric proteins in the gas phase has been extended to assemblies containing more than one protein. Ion mobility measurements of complex macromolecular protein complexes at atmospheric pressure [8] or at a pressures of ca. 1 ϫ 10 Ϫ3 Torr have shown some success [9,10]. The resolution of these experiments, ca. 5-20, is lower than more conventional mobility measurements at pressures of a few Torr.Protein-protein and protein-ligand complexes can be studied by gas-phase H/Dx of trapped ions. The pressures of deuterating reagent in linear trap experiments can be ϳ5 ϫ 10 Ϫ3 Torr, ca. 10 3 times greater than used in 3D traps [20], 10 1 to 10 2 times greater than used in mobility experiments [26,27], and 10 3 to 10 4 times greater than in ICR experiments [21][22][2...

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“…Occasionally, this was complemented by CD and fluorescence spectroscopic measurements [27,28]. Other studies were based on measurements of haptoglobin binding [29], autooxidation kinetics [30], radioactive labeling [31], as well as stopped-flow pH measurements [32]. The in vitro assembly of Hb has most commonly been triggered by mixing initially separated ␣ h and ␤ h subunits under native solvent conditions.…”

mentioning

confidence: 99%

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

Boys

Konermann

2007

J. Am. Soc. Mass Spectrom.

101

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The native structure of hemoglobin (Hb) comprises two ␣-and two ␤-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded ␣-and ␤-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric ␣-and ␤-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded ␣-and ␤-globin. However, some unexpected features are observed, e.g., a heme-deficient ␤-globin dimer that represents an off-pathway intermediate. Monomeric ␤-globin is capable of binding heme before forming a complex with an ␣-subunit. This observation suggests that holo-␣-apo-␤ globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes. (J Am Soc Mass Spectrom 2007, 18, 8 -16)

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Tetramer‐dimer equilibrium of oxyhemoglobin mutants determined from auto‐oxidation rates

Cited by 53 publications

References 64 publications

Low NO Concentration Dependence of Reductive Nitrosylation Reaction of Hemoglobin

Low NO Concentration Dependence of Reductive Nitrosylation Reaction of Hemoglobin

Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

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