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

Abstract

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...

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

“…Earlier studies have shown that the inhibitory effect of HbF on the polymerization of deoxy-HbS is dependent on the formation of heterotetramers (␣ 2 ␤ S ␥) (37). A similar mechanism was proposed to explain the polymerization inhibitory effects of the naturally occurring minor hemoglobin, HbA 2 (␣ 2 ␦ 2 ) (38). Unlike the ␣ 2 ␤ A ␤ S heterotetramer, the ␣ 2 ␤ S ␥ and ␣␤ S ␦ heterotetramers are excluded from the sickle polymer, which accounts for the increased inhibitory effects of HbF and HbA 2 relative to HbA.…”

Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry

“…Key terms: Image analysis, sickle cell disease, hemoglobin F Sickle cell disease (SCD) is caused by a genetic disorder of hemoglobin ( 17), sickle hemoglobin (Hb S), which polymerizes under hypoxic conditions (12). It is known that fetal hemoglobin (Hb F) is an effective inhibitor of this polymerization (1,10, 22,27) and that Hb F level is an important determinant of the clinical course of patients with this disease. Patients with higher Hb F levels generally have a much milder clinical course, and Platt et al (25) recently reported that mean pain rate decreases as mean Hb F level increases with no threshold when hundreds of data were analyzed.…”

Hemoglobin Variants: Biochemical Properties and Clinical Correlates

“…Hb D-Ibadan (b87 [F3] Thr . Lys), which introduces a lysine residue at the b87 position, is presumed to have decreased interaction with the mutant Val residue at HbS b6 (WatsonWilliams et al 1965;Nagel et al 1979). Thus, Hb D-Ibadan inhibits HbS polymerization.…”

“…Several other Hb variants modulate the severity of sickle cell anemia (see also Cao and Kan 2012;Schechter and Elion 2012;Serjeant and Rodgers 2012). For example, g globin inhibits polymerization of HbS (Nagel et al 1979). This effect is attributable to differences in several amino acid residues compared with the corresponding b chain, including g80 and g87 (Adachi and Asakura 1979;Nagel et al 1979;Adachi et al 1996).…”

“…For example, g globin inhibits polymerization of HbS (Nagel et al 1979). This effect is attributable to differences in several amino acid residues compared with the corresponding b chain, including g80 and g87 (Adachi and Asakura 1979;Nagel et al 1979;Adachi et al 1996). Hb D-Ibadan (b87 [F3] Thr .…”