Gelation of sickle cell hemoglobin IV. Phase transitions in hemoglobin S gels: Separate measures of aggregation and solution-gel equilibrium

Abstract

Two assays of equilibrium properties in the gelation of deoxyhemoglobin S were carried out by analytical ultracentrifugation on the same sample: C sat, the monomer concentration in equilibrium with the fully formed gel, was obtained as the supernatant concentration after sedimentation of a preformed gel. The presence of a plateau region during sedimentation of the supernatant and the rate of sedimentation of the boundary from which C sat was measured indicate that centrifugation did not alter the pre-existing equilibrium and that the supernatant consisted of monomers. The centrifugation was then continued to equilibrium to obtain a distribution showing a sharp increase in molecular weight at C agg, the monomer concentration at which a small amount of polymerization to large aggregates begins. The primary result is that C sat > C agg under all conditions. The different values of the two parameters indicate that they reflect two separate transitions and that the overall monomer to gel process has a limited co-operativity. Within the limits of the method C sat is independent of total hemoglobin concentration. The two transitions divide the overall range of total hemoglobin concentration into an essentially monomeric region at concentrations below C agg, a region in which isotropically oriented polymers exist, occurring when monomer concentration lies between C agg and C sat, and a two-phase region of conjugate isotropic and anisotropic phases when monomer concentration equals C sat. These regions correspond to zones in the ultracentrifuge equilibrium distribution. In this scheme C agg depends only on the interaction energy of polymerization. C sat depends on entropic factors which induce tactoid formation as well. C sat, while a monomer concentration, reflects a saturation not of monomers in relation to a polymeric phase, but of polymers in the isotropic phase in relation to the anisotropic or tactoidal polymerized phase. As such, C sat represents a supersaturated state of isolated monomers. The ratio C sat C agg = 1.23 in stripped hemoglobin ‡ ‡ Stripped indicates hemoglobin in the absence of organic phosphates. and equilibrium distributions in the zone of isotropically oriented polymers were both used to obtain an order of magnitude estimate of polymer size, found to be much smaller than that of hemoglobin S fibers. This further confirms that gelation does not consist of a single transition and phase change with near infinite co-operativity of polymerization. C sat as well as C agg are lowered by 2,3,diphosphoglycerate and inositol hexa-phosphate. Decreasing pH near 7 also favors gelation; in stripped hemoglobin a pH optimum for gelation occurs near pH 6.8. The apparent van't Hoff ΔH for stripped hemoglobin is about 3 kcal/mol for C sat and 2 kcal/mol for C agg.