[37] Measurement and analysis of ligand-linked subunit dissociation equilibria in human hemoglobins

Turner, Benjamin W.; Pettigrew, Donald W.; Ackers, Gary K.

doi:10.1016/0076-6879(81)76147-0

Cited by 70 publications

(49 citation statements)

References 26 publications

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“…Dimer-tetramer equilibrium constants for normal hemoglobin Ao ligated with CO and for Mn3+ hemoglobin (species 41) were determined directly by analytical gel chromatography (15). The resulting value of 4K21, in combination with the dissociation rate constant from the haptoglobin experiment, provides a determination of the association rate constant for species 41 in agreement with that obtained kinetically.…”

Section: Methodssupporting

confidence: 60%

“…The allosteric Monod-Wyman-Changeux (MWC) model provides a rigorous theoretical treatment for the simplest mechanism of cooperative switching in a tetrameric system that has only two molecular structures (17 Theoretical treatment of this problem (9) provides the fundamental relationship K= K'R (1 + LcO. 15] Here iK2 is the intrinsic equilibrium constant for dimertetramer assembly for a tetramer with i ligated subunits (i = 0, 1, 2, 3, 4), and K2R is the intrinsic assembly constant for forming an R-state tetramer from two dimers. The allosteric constant, L, represents the equilibrium between unligated conformers (R and T) while c is the ratio of their affinities for ligand (see ref.…”

Section: Resultsmentioning

confidence: 99%

“…In an extensive series of studies of normal hemoglobin over a wide range of pH values (8) and with mutant or chemically modified hemoglobins, including hybrids (5,15), the value of the assembly rate constant remained unchanged.…”

Section: Methodsmentioning

confidence: 99%

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Three-state combinatorial switching in hemoglobin tetramers: comparison between functional energetics and molecular structures.

Smith

Gingrich

Hoffman

et al. 1987

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

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In a previous study on cyanomethemoglobin the 10 tetrameric species (each with a unique combination of ligated and unligated subunits) were found to exhibit three distinct free energies of cooperative interaction. The distribution of these free energies among the partially ligated species is incompatible with a two-state mechanism of molecular switching and requires a minimum of three molecular structures with distinctly different free energies of heme-heme interaction. Ligand-linked transitions between the three cooperativity states were found to be "combinatorial"-i.e., dependent upon changes in both the number and specific configuration of bound ligands. Here we present results from two other chemical systems that mimic intermediate oxygenation states. In these systems the heme iron is replaced by manganese in certain of the subunits. We find the same distribution of cooperative free energies as reported for the cyanomethemoglobin system. These results demonstrate that the three-state combinatorial nature of cooperative switching is neither a special feature of the cyanomet reactions nor of the substitution of manganese for iron, but reflects a fundamental property of hemoglobin. These findings are compared with crystallographic structural results on partially ligated hemoglobins. (Fig. 2). In view of the potential significance of these findings to the mechanism of hemoglobin cooperativity, it is essential to determine whether three-state combinatorial switching is a special property of the cyanomet reactions with hemoglobin and whether the three levels of cooperativity correspond to distinct molecular structures. In the present study we determined the cooperative free energies for partially ligated tetramers in two additional, chemically different, systems that mimic partially ligated tetramers. The species we have studied are depicted in Fig. 3: in system A the hemes are replaced in some of the subunits by Mn2+ protoporphyrin IX, providing a functional analog of unligated subunits (2, 14) while the remaining subunits have normal hemes that are ligated with carbon monoxide (CO); in system B, the unligated subunits contain normal heme while the ligated subunits contain hemes replaced by Mn3+ protoporphyrin IX. 01Here we analyze the functional energetics of these systems in terms of the minimum number of molecular structures required. We will also compare the energetics with crystallographic results (3, 4). AG, = AGi -iAG~,where AGQ is the standard Gibbs free energy for reacting i moles of ligand with a tetramer and AG. is the intrinsic free energy per site for the same reaction in the absence of cooperativity (5). In hemoglobin tetramers iAGx generally has a larger negative value (higher affinity) than AG, so that AGc is positive. A finding that AGc = 3.0 kcal (1 cal = 4.18 J) for Abbreviation: MWC, Monod-Wyman-Changeux. 7089The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §173...

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Section: Methodssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Three-state combinatorial switching in hemoglobin tetramers: comparison between functional energetics and molecular structures.

Smith

Gingrich

Hoffman

et al. 1987

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

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“…For example, whereas the dimer-tetramer dissociation constant for normal human HbA is about lo-' M in its oxygenated state and M in its deoxygenated form (Turner et al, 1981), the corresponding values for the natural mutants at Asp%(@) are increased by 3-5 orders of magnitude (Turner et al, 1981Doyle et al, 1992). We have measured the extent of dimer formation of the oxygenated D99K mutant Hb by several procedures including gel filtration, haptoglobin binding of ~$3 dimers determined by filtration of the haptoglobin-Hb dimer complex and by quenching of fluorescence, and by light scattering, as described below.…”

Section: Dissociation Of the Mutant Hbmentioning

confidence: 99%

Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

Yanase¹,

Cahill²,

Llano³

et al. 1994

Protein Science

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Site-directed mutagenesis of an important subunit contact site, , by a Lys residue (D99K(@)) was proven by sequencing the entire @-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(@) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the a-and @-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(@) recombinant mutant forms have differences in their heme-protein environments.

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“…This equilibrium exists under physiological conditions, is an important determinant of hemoglobin function, and shifts depending on various conditions such as pH, ionic strength, and Hb concentration. Subunit dissociation to dimers upon dilution has been studied by a variety of methods, all of which involve measurement of the average molecular weight of Hb dimer and tetramer (16). Our present results show that in the presence of excess ␣ chains, heterodimers rather than heterotetramers form because of the low concentration of newly synthesized globin chains generated in this cell-free system (ϳ10 Ϫ8 M) compared with the dissociation constant (K d ) of tetrameric Hb (ϳ10 Ϫ6 M) (16 -18).…”

Section: Synthesis Of Radiolabeled ␥-Or ␤-Globinmentioning

confidence: 99%

Assembly of Human Hemoglobin (Hb) β- and γ-Globin Chains Expressed in a Cell-free System with α-Globin Chains to Form Hb A and Hb F

Adachi¹,

Zhao²,

Surrey³

2002

Journal of Biological Chemistry

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Rates of in vitro synthesis of radiolabeled ␥ and ␤ chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled ␤ or ␥ chains at the start of chain synthesis generated radiolabeled ␤ 4 or ␥ 2 and ␥ 4 chains, respectively. If unlabeled ␣-globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled ␣␤ or ␣␥ bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled ␣␤ or ␣␥ prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If ␣ chains were added after synthesis of radiolabeled ␥ chains made in the presence of unlabeled ␥ chains, then little radiolabeled ␣␥ formed. In contrast, if ␣ chains were added after synthesis of radiolabeled ␤ chains made in the presence of unlabeled ␤ chains, then radiolabeled ␣ 2 ␤ 2 formed. These findings suggest that ␤ and ␥ chains associate with ␣ chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable ␥ 2 dimers and suggests that ␣ chains may bind to nascent non-␣ chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo.Assembly in vitro of human Hb 1 subunits (␣ and non-␣ chains) into stable Hb heterotetramers (e.g. ␣ 2 ␤ 2 or ␣ 2 ␥ 2 ) using purified globin chains has been explored and a 3-step mechanism proposed (1-8). The ␣ chains are in monomer/dimer equilibrium-favoring monomers, whereas non-␣ chains are in monomer/tetramer equilibrium-favoring tetramers (9, 10). It is generally assumed that dissociation of these oligomeric subunits into monomers must occur before these two different chains can combine to form ␣␤ or ␣␥ dimers, which then associate to form tetrameric Hb (␣ 2 ␤ 2 or ␣ 2 ␥ 2 ) (11, 12). In addition, the assembly of ␣␤ or ␣␥ dimer was postulated to be the rate-limiting step for assembly in vivo and has been theorized to be governed by electrostatic attractions between ␣-and non-␣ partner subunits (8, 12). Furthermore, from in vitro studies it is known that Hb F formation using purified ␥-and ␣-globin chains is very slow compared with Hb A using purified ␤-and ␣-globin chains (11). In fact, our previous studies showed approximately a 10 5 -fold slower rate of assembly in vitro for Hb F compared with Hb A (11). The slow rate of Hb F formation in vitro is caused by stable ␥ 2 dimer formation and is unlikely to occur in erythroid precursors (11). We also found that even at low concentrations (Ͻ5 M), ␥ chain dimers do not dissociate readily into monomers, resulting in decreased assembly with ␣ chains. Furthermore, our results showed that the assembly of [Ile 116 3 His]␥ chains with ␣ chains was similar to that of ␤ chains, whereas the assembly of [Thr 112 3 Cys]␥ with ␣ chains was similar to wild type ␥ chains (11). These findings indicate that amino acid differences between [Ile 116 ]␥ and [His 116 ]␤ at ␣ 1 ␥ 1 and ␣ 1 ␤ 1 interaction sites, respectively, a...

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[37] Measurement and analysis of ligand-linked subunit dissociation equilibria in human hemoglobins

Cited by 70 publications

References 26 publications

Three-state combinatorial switching in hemoglobin tetramers: comparison between functional energetics and molecular structures.

Three-state combinatorial switching in hemoglobin tetramers: comparison between functional energetics and molecular structures.

Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

Assembly of Human Hemoglobin (Hb) β- and γ-Globin Chains Expressed in a Cell-free System with α-Globin Chains to Form Hb A and Hb F

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