Solubilization of hemoglobin S by other hemoglobins.

Abstract

The polymerization of mixtures of Hb S with hemoglobins A, A2, and F has been investigated by analysis of the proportions of S and non-S hemoglobin both in the supernate and-in the pellet after centrifugation. In all cases the non-S hemoglobin was incorporated into the polymer even in the absence of hybrids in the order A > A2 > F. The solubility of Hb S is substantially increased by the other hemoglobins, especially by Hb F. which would account for its antisickling effect. It appears that the excluded volume effect of the other hemoglobin on Hb S is largely counterbalanced by the solubilizing effect arising from the interaction between the two hemoglobins in solution. The ability of hybrid hemoglobins to gel was demonstrated directly with tetramers in which afts dimers were covalently linked to aflA, a5A2, and ayF dimers.The so-called "sickle-sparing" effect of other hemoglobins on hemoglobin S (Hb S) was described more than 20 years ago by Singer and Singer (1) and by Allison (2), who found that some hemoglobins such as Hb A and Hb C greatly decrease the concentration of Hb S necessary for gelling, whereas Hb F had a much smaller effect. It was assumed by these authors and in much of the subsequent work with other hemoglobins (3-6) that the "sickle-sparing" hemoglobins act by substituting for Hb S in the polymer, i.e., by copolymerizing with it. By contrast, it was concluded that Hb F and, more recently, Hb A2 (7-9) do not copolymerize with Hb S.However, it has now been realized that another factor plays a major role in the gelling of Hb S in the presence of other hemoglobins (10-12). In these very concentrated and nonideal solutions the presence of any other hemoglobin [and, indeed, any The purpose of the work reported here was therefore to investigate the participation of Hb F and Hb A2 in the polymerization of Hb S by direct analysis, not only of the supernate, but also of the gel. In addition, hemoglobins in which an af3s dimer was covalently crosslinked to either an acy or an a6 dimer were used to test the ability of these mixed tetramers to polymerize. MATERIALS AND METHODSThe hemoglobins were isolated from hemolysates, prepared as described (19) from normal blood, sickle trait blood, and cord blood. After dialysis against 0.1 M glycine at pH 7.6, the hemolysates were fractionated on DEAE-cellulose, Whatman DE52 (20), under the conditions shown in Table 1. Sectional columns (Kontes Glass) were used and the desired hemoglobin was removed from the appropriate segment without elution of the column (21).Crosslinked (XL) hemoglobins were prepared by using the reagent 2-nor-2-formylpyridoxal 5'-phosphate as described (22,23). For the preparation of crosslinked mixed tetramers, the reaction was performed on equimolar amounts of Hb S and the other hemoglobin that had been mixed under oxygen to allow for hybridization. After the coupling reaction, which takes place under anaerobic conditions, the hemoglobins were converted to the cyanmet form by oxidation with 1.2 equivalents of potassium ferricyanide...

Polymerization of Three Hemoglobin A2 Variants Containing Valδ6 and Inhibition of Hemoglobin S Polymerization by Hemoglobin A2

“…The 0.1-unit difference in left shift on the X axis of the line for A 2 S mixtures compared with that of FS mixtures in the experiments in a high phosphate buffer (Fig. 7) indicates that Hb A 2 inhibits Hb S polymerization slightly less than Hb F, which corresponds to results in 0.1 M phosphate buffer reported previously by Benesch et al (9). These differences can be explained by participation of some ␣ 2 ␤ S ␦ hybrids in the formation of nuclei with Hb S at about one-eighth the efficiency of Hb S. Copolymerization with Hb S occurs because Thr ␤87 in ␤ S in the ␣ 2 ␤ S ␦ hybrids can interact with Val ␤6 in Hb S. In contrast, ␣ 2 ␤ S ␥ hybrids in FS mixtures are completely excluded from Hb S nucleation.…”

“…Mixtures of Hb A 2 and Hb S, like mixtures of Hb F and Hb S, have a higher solubility than Hb S alone and result in inhibition of Hb S polymerization (1,2). The primary inhibitory effects of Hb A 2 and Hb F in these mixtures are to exclude the asymmetrical ␣ 2 ␤ S ␦ and ␣ 2 ␤ S ␥ hybrids, respectively, as well as Hb A 2 and Hb F from initiation of polymerization with Hb S (3)(4)(5)(6)(7)(8)(9)(10)(11). Studies comparing minimum gel concentrations for naturally occurring hemoglobin variants, including the Lepore (␦␤ hybrid) hemoglobins, suggested that Gln ␦87 and Ala ␦22 in Hb A 2 are important sites potentiating inhibition of polymerization of deoxy Hb S (5).…”

Direct observation of photolysis-induced tertiary structural changes in hemoglobin

“…Several hybrid Hbs of this kind have been used as a model system to study T-state Hb (28)(29)(30)(31)(32). The other approach to further improving crystal quality is to use intramolecular crosslinking, which prevents the dissociation of Hb tetramer into ␣␤ dimers (33)(34)(35). It has been shown that dissociation into ␣␤ dimers is an obligatory step in thermal unfolding (36) and dissociation of heme from globin (37), both of which may interfere with crystallization (38).…”

Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin.

“…One of the monofunctional aspirin-based derivatives, however, also has a low oxygen affinity but does not require crosslinking (10). Several of the bifunctional reagents, such as 2-nor-2-formyl-5-pyridoxal 5'-phosphate (2,11), bispyridoxal tetraphosphate (12), bis(3,5-dibromosalicyl)fumarate (13), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) (14), yield a crosslinked hemoglobin derivative with a low oxygen affinity when the crosslinker is used with deoxyhemoglobin. In general, the reduced oxygen affinity of a hemoglobin derivative together with its inability to dissociate form the basis for its consideration as a blood substitute.…”

“…Some crosslinked hemoglobin derivatives have a Mr of 64,000-i.e., crosslinking only within a given tetramer (12)(13)(14)(15), and others have Mr values of 128,000 or greater-i.e., crosslinking between hemoglobin tetramers (16,17). The latter derivatives include the dextran-hemoglobin complexes (18), hemoglobin conjugates with polyethylene glycol (19), and glutaraldehyde-treated pyridoxylated hemoglobin, which are very large polymers of different molecular weights.…”