Assembly of Oxyhemoglobin from Isolated α and β Chains

Kawamura, Yasuko; Nakamura, Satoshi

doi:10.1093/oxfordjournals.jbchem.a134241

Cited by 14 publications

(6 citation statements)

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“…Interaction of ␣ and ␤ chain monomers in Reaction III is very rapid compared with dissociation of ␤ 4 tetramers to monomers (7). The second-order rate constant of Reaction III for ␣ and ␤ A ( ␣␤ k 5 ) is about 10 5 M Ϫ1 s Ϫ1 (20,26), which means that ␣ monomer binds monomeric ␤ A chain very rapidly, roughly following second-order reaction kinetics. In contrast, dissociation of ␤ 4 34␤ is very slow exhibiting a dissociation constant ( ␤ k 4 ) of about 10 Ϫ3 s Ϫ1 (20).…”

Section: ␤112mentioning

confidence: 99%

“…The second-order rate constant of Reaction III for ␣ and ␤ A ( ␣␤ k 5 ) is about 10 5 M Ϫ1 s Ϫ1 (20,26), which means that ␣ monomer binds monomeric ␤ A chain very rapidly, roughly following second-order reaction kinetics. In contrast, dissociation of ␤ 4 34␤ is very slow exhibiting a dissociation constant ( ␤ k 4 ) of about 10 Ϫ3 s Ϫ1 (20). These results indicate that isolated single ␣ and ␤ chains assemble quickly but that formation of tetrameric hemoglobin depends on ␤ 4 stability and total amounts of monomer in solution.…”

Section: ␤112mentioning

confidence: 99%

“…␤ 4 tetramer and ␤-monomer levels for each of the ␤ chain variants were assessed by size-exclusion chromatography (17,20,21). ␤ A -Globin chains in solution exist as homo-tetramers (␤ 4 ) rather than monomers in the absence of ␣-globin chains (1).…”

Section: Expression and Purification Of Soluble ␤-Globinmentioning

confidence: 99%

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Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Yamaguchi¹,

Pang²,

Reddy³

et al. 1998

Journal of Biological Chemistry

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In order to assess the role of ␤112 Cys in homo-and hetero-tetrameric hemoglobin formation, we expressed four ␤112 variants (␤ 112Cys3 Asp , ␤ 112Cys3 Ser , ␤ 112Cys3 Thr , and ␤ 112Cys3 Val ) and studied assembly with ␣ chains in vitro. ␤112 Cys is normally present at ␤ 1 ␤ 2 and ␣ 1 ␤ 1 interaction sites in homo-(␤ 4 ) and hetero-tetramers (␣ 2 ␤ 2 ). ␤ 4 formation in vitro was influenced by the amino acid at ␤112. ␤112 Asp completely inhibited formation of homo-tetramers, whereas ␤112 Ser showed only slight inhibition. In contrast, ␤112 Thr or Val enhanced homotetramer formation compared with ␤ A chains. Association constants for homo-tetramer formation increased in the order of ␤ 112Cys3 Ser , ␤ A , ␤ 112Cys3 Thr , and ␤ 112Cys3 Val , whereas the value for ␤ 112Cys3 Asp was zero under the same conditions. These ␤112 changes also affected in vitro ␣ 2 ␤ 2 hetero-tetramer formation. Order of ␣ 2 ␤ 2 formation under limiting ␣-globin chain conditions showed Hb ␤C112S > Hb A > Hb S ‫؍‬ Hb ␤C112T ‫؍‬ Hb ␤C112V > > > Hb ␤C112D. Hb ␤112D can form tetrameric hemoglobin, but this ␤112 change promotes dissociation into ␣ and ␤ chains instead of ␣␤ dimer formation upon dilution. These results indicate that amino acids at ␣ 1 ␤ 1 interaction sites such as ␤112 on the G helix play a key role in stable ␣␤ dimer formation. Our findings suggest, in addition to electrostatic interaction between ␣ and ␤ chains, that dissociation of ␤ 4 homo-tetramers to monomers and hydrophobic interactions of the ␤112 amino acid with ␣ chains governs stable ␣ 1 ␤ 1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.Equimolar amounts of ␣-and ␤-globin chains of human hemoglobin self assemble to form ␣ 2 ␤ 2 tetramer. In addition, isolated ␤ chains also assemble to form ␤ 4 homo-tetramers (1). Extensive previous studies using naturally occurring variants and our recent studies using recombinant ␤ chain variants showed that affinity between ␣ and ␤ chains is promoted by negatively charged ␤ chains and is independent of charge location on the surface except at the ␣ 1 ␤ 1 interaction site (2-5). Our previous studies showed that affinity is promoted by negatively charged ␤ chains up to a maximum of two additional net negative charges (5). In addition, we showed that ␤112 Cys located at an ␣ 1 ␤ 1 interaction site on the G helix is critical for facilitating formation of stable ␣␤ dimers which then form functional hemoglobin tetramers. We also demonstrated that ␤ 112Cys3 Asp inhibits formation of stable ␣ 1 ␤ 1 and ␤ 1 ␤ 2 interactions in ␣ 2 ␤ 2 and ␤ 4 tetramers, respectively (5).X-ray diffraction analysis of ␤ 4 and ␣ 2 ␤ 2 showed that ␤112 Cys on the G helix is involved in ␤ 1 ␤ 2 and ␣ 1 ␤ 1 subunit interactions, respectively, and that there is similarity between the quaternary structure of carbonmonoxy ␤ 4 and carbonmonoxy ␣ 2 ␤ 2 tetramers (6). The detailed role of ␣ 1 ␤ 1 interaction s...

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Section: ␤112mentioning

confidence: 99%

Section: ␤112mentioning

confidence: 99%

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Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Yamaguchi¹,

Pang²,

Reddy³

et al. 1998

Journal of Biological Chemistry

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“…The Soret CD of hemoglobin is ascribed to the interaction between the heme and the surrounding aromatic amino acid residues (Hsu and Woody 1971). The interaction is provided not only by the heme binding to each chain but als by c~1/~1 contacts (Kawamura et al 1982;Kawamura and Nakamura 1983;Mawatari et al 1983). It is therefore plausible that the ligation of heme to the proximal His would cause significant conformational changes of the chains, resulting in an increase in the el/~l interaction.…”

Section: Third Step" the Ligand Replacement Of Cnby The Proximal Hismentioning

confidence: 99%

Kinetics of the reconstitution of hemoglobin from semihemoglobins ? and ? with heme

et al. 1992

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Kinetics of the reconstitution of hemoglobin from semihemoglobins alpha and beta with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins alpha and beta are occupied by heme in the alpha and beta chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin alpha, the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin beta changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin alpha retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin beta.

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“…The newly synthesized o: and fl monomers associate to form dimer intermediates which aggregate to form the o~2/32 tetramer. In vitro subunit competition (Shaeffer, 1980;Mrabet et al, 1986) and kinetic (Kawamura and Nakamura, 1983;McDonald et al, 1987;Joshi and McDonald, 1994) Biochemistry Program, Department of Chemistry, College of Arts and Sciences, University of Massachusetts at Lowell, Lowell, Massachusettg 01854..…”

Section: Introductionmentioning

confidence: 99%

Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence

O'Malley

McDonald

1994

J Protein Chem

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A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14(A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104(G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human alpha-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer, pH 7.0, at 5 degrees C, and the structural flexibility of alpha-apohemoglobin, in the absence and presence of human beta-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (EQ) and Förster distances (R0Q) were analyzed by least-squares to determine the goodness of fit (chi R2) for the assumed distribution parameters: average distance r and half-width hw. Data for alpha-apohemoglobin in the absence and presence of beta h chains yielded values for r of 18 and 22 A and hw of 20 and 8.5 A, respectively. Although the increase in r for alpha-apohemoglobin in the presence of beta h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the alpha-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.

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Assembly of Oxyhemoglobin from Isolated α and β Chains

Cited by 14 publications

References 0 publications

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Kinetics of the reconstitution of hemoglobin from semihemoglobins ? and ? with heme

Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence

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