Polymerisation of haemoglobin SA hybrid tetramers

Bookchin, Robert M.; Balazs, Tania; Nagel, Ronald L.; Tellez, I.

doi:10.1038/269526a0

Cited by 46 publications

(19 citation statements)

References 9 publications

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“…More recently, it has been shown that this inhibition depends on the formation, in Hb S/Hb F mixtures, of asymmetrical hybrid tetramers of the type a2lS3y. Such an inhibitory effect did not occur with the formation of a2t3S#fA hybrids (4,5). Thus, the replacement of one of the Os chains of Hb S by a y chain appeared to have a strong inhibitory effect on polymerization.…”

mentioning

confidence: 80%

Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.

Nagel¹,

Bookchin²,

Johnson³

et al. 1979

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

187

106

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We have previously found that the inhibitory effect of hemoglobin F (Hb F) on the polymerization of Hb S proceeds via the formation of asymmetrical hybrid tetramers of the type a2ts. Examination of the gelling properties of binary mixtures of Hb S and several Hb variants now shows that, among the y chain amino acid residues that differ from those of the P chain, residues y80 (EF4) and 'y87 (F3) are at least partly responsible for this inhibition. Furthermore, we find that mixing Hb A2(a2h2) with Hb S strongly inhibits gelling to an extent similar to that seen with Hb S/Hb F mixtures; this inhibition is attributable to amino acid differences between the 5 and P chain sequences at positions 522 (B4) and 587 (F3). Therefore, residues 22, 80, and 87 of the f chain appear to be involved in intermolecular contact sites that stabilize the deoxy Hb S polymers. The capacity of some Hb variants to facilitate or impair the polymerization of Hb S is now well known (1). It has long been recognized that these effects correlate with the clinical severity of genetic variants of sickle cell disease in which two major Hb components coexist in the erythrocytes (2). Our laboratory has developed the use of binary Hb mixtures (Hb X + Hb S) to map the residues involved in the interactions between Hb S tetramers in the polymer (3). The inhibitory effect of Hb F on the polymerization of Hb S has been well documented (1). More recently, it has been shown that this inhibition depends on the formation, in Hb S/Hb F mixtures, of asymmetrical hybrid tetramers of the type a2lS3y. Such an inhibitory effect did not occur with the formation of a2t3S#fA hybrids (4,5). Thus, the replacement of one of the Os chains of Hb S by a y chain appeared to have a strong inhibitory effect on polymerization. In the present article we will define part of the structural basis of this inhibition. In addition, we report a prominant inhibitory effect of Hb A2, on polymerization in mixtures with Hb S and provide data concerning the structural basis of this inhibition. METHODSHemolysates were made from blood' samples obtained after informed consent from donors with sickle cell anemia (SS) documented in our Heredity Clinic. The erythrocytes from these donors contained less than 5% (mean) Hb F and were used without further purification. (MGCs) were determined at 240C as described (6). It should be noted that we perform this assay in a manner such that the MGC values closely approximate the values we obtain by measuring the supernatant Hb concentration after highspeed centrifugation of the gel: the Hb mixture is fully deoxygenated for at least 40 min before its concentration is allowed to rise very slowly (by the evaporation of the solvent), and the determination is considered reliable only if the final Hb concentration is less than 2 g/dl higher than the starting concentration and if the total duration of the procedure from the time evaporation is begun is at least 30 min but less than 2 hr. For each mixture, repeat MGC determinations differed from the mean...

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mentioning

confidence: 80%

Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.

Nagel¹,

Bookchin²,

Johnson³

et al. 1979

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

187

106

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“…Our preliminary measurements of C e using a new method [4] gave a value of 59.1 _+ 0.9 g/dl, nearly 20% below the previous estimates of =69 g/dl. Those results prompted us to assess the role of polymerassociated water in the cell volume changes accompanying Hb polymerization by comparing predictions from models with different values of C e. We first compared predictions from a model with Cp = 69 g/dl (Table 1 and Fig.…”

Section: Role Of Polymer Hydration On the Osmotic Effects Of Polymerimentioning

confidence: 83%

Osmotic effects of protein polymerization: Analysis of volume changes in sickle cell anemia red cells following deoxy-hemoglobin S polymerization

Lew

Bookchin

1991

J. Membrain Biol.

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Polymerization-depolymerization of proteins within cells and subcellular organelles may have powerful osmotic effects. As a model to study these we analyzed the predicted volume changes following hemoglobin (Hb) S polymerization in sickle cell anemia (SS) red cells with different initial volumes. The theoretical analysis predicted that dehydrated SS red cells may sustain large polymerization-induced volume shifts whose direction would depend on whether or not small solutes were excluded from polymer-associated water. Experiments with SS cells from promptly fractionated venous blood showed oxygenation-induced swelling, maximal in the densest cells, in support of nonexclusion models. The predicted extent of cell dehydration on polymerization was strongly influenced by factors such as the dilution of residual soluble Hb and the increased osmotic contribution of Hb in cells dehydrated by salt loss, largely overlooked in the past. The osmotic effects of polymer formation may thus play an important part in microcirculatory infarction by dense SS cells, as they become even denser and stiffer during deoxygenation in the capillaries.

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“…In the current scenario, the definition of a fiber contact by both theoretical as well as experimental approach is centered on specific amino acid residues. The solution studies consider a given residue as a fiber contact only if the mutation of that site results in an altered polymerization behavior relative to the native HbS (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). On the other hand, the fiber models define a contact residue based on the distance; residues with contact distances of about 5 Å or less are generally considered as interacting partners (26,27).…”

Section: Discussionmentioning

confidence: 99%

“…Deoxygenated sickle hemoglobin (HbS) polymerizes into long helical fibers that are believed to be responsible for the pathophysiology of the sickle cell disease. The knowledge gleaned so far from structural analysis of HbS crystal (2)(3)(4), solution polymerization studies of natural variants or engineered mutant hemoglobins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), and electron microscopic studies (25) have led to a 14-stranded model of the fiber (26,27). These strands appear as seven double strands of the type found in HbS crystals, albeit with a slight twist caused by fiber packing.…”

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confidence: 99%

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

Sudha

Anantharaman

Sivaram

et al. 2004

Journal of Biological Chemistry

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The AB and GH regions of the ␣-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala 19 and Ala 21 of the AB corner with more flexible Gly or rigid ␣-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of ␣-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (␣Asn 78 3 His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of ␣Gln 78 , His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (␣Gln 78 3 His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His 78 . Similar synergistic coupling, between ␣His 78and an intra-double-strand contact point (␣16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with ␣78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.Sickle cell anemia arises because of a point mutation at the sixth position of the ␤-chain (␤Glu 6 3 Val) in the hemoglobin (Hb) 1 molecule (1). Deoxygenated sickle hemoglobin (HbS) polymerizes into long helical fibers that are believed to be responsible for the pathophysiology of the sickle cell disease. The knowledge gleaned so far from structural analysis of HbS crystal (2-4), solution polymerization studies of natural variants or engineered mutant hemoglobins (5-24), and electron microscopic studies (25) have led to a 14-stranded model of the fiber (26, 27). These strands appear as seven double strands of the type found in HbS crystals, albeit with a slight twist caused by fiber packing. The models specify several amino acid residues from both ␣-and ␤-chains that participate in inter-or intradouble-strand contacts stabilizing the fiber structure. By and large, the fiber models agree with the solution polymerization experiments of mutant hemoglobins in that polymerizationsensitive mutations are usually located in fiber contact regions (27). However, the models present an approximate...

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Polymerisation of haemoglobin SA hybrid tetramers

Cited by 46 publications

References 9 publications

Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.

Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.

Osmotic effects of protein polymerization: Analysis of volume changes in sickle cell anemia red cells following deoxy-hemoglobin S polymerization

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

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