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Solubilities of deoxygenated sickle cell hemoglobin (deoxy-Hb S), at varying pH and temperature over a range of concentrations encompassing those found in erythrocytes, were measured. The technique involved ultracentrifugation, which gave values of the supernatant concentration and the mass of the sedimented material. The data establish that the solubility of deoxy-Hb S is the saturation concentration and is independent of initial concentration. The mass of the pellet phase increases linearly with initial concentration. Moreover, the saturation concentration represents the critical concentration above which monomers are in equilibrium with polymers. These polymers are the putative cause of erythrocyte deformation associated with sickle cell anemia. The solubility-pH profiles of deoxy-Hb S at various temperatures, unlike those of other proteins, show no minima at the isoelectric pH, but instead snow a marked decrease in solubility below pH 7.0, indicating the predominance of polymerization over the expected increase in solubility. Deoxy-Hb S, within specified ranges of temperature and pH, possesses a negative temperature coefficient of solubility, a property characteristic of hydrophobic interactions. The saturation concentration is, however, temperature independent at conditions close to physiological. The enthalpy of polymerization (3.5 kcal/mol) is temperature independent from 60 to 220 for all pH values between 6.45 and 7.40. In the range of 220 to 38°, this parameter becomes less endothermic, having a value of 2.5 kcal/mol at pH 6.45 and a value of zero at pH 7.20. Such behavior of the system suggests a phase transition near 220. Within the range of.conditions examined the polymerization is entropically driven.Sickle cell hemoglobin (Hb S) is a variant of normal adult hemoglobin (Hb A) in which a substitution of valine for glutamic acid occurs at position 6 in both ,3 chains. Deoxygenated solutions of Hb S, at concentrations comparable to those within erythrocytes, form liquid crystals or tactoids. These nematic crystals of deoxy-Hb S, the putative cause of erythrocyte sickling, have been characterized optically by their birefringence and polarization dichroism (1, 2). Structural information derived from studies of both x-ray diffraction and optical diffraction of electron micrographs has provided evidence that these paracrystalline arrays are composed of helical microtubules packed into square lattices (3, 4).The term lowest gelling point has been used (5) to denote the concentration at which a solution of deoxy-Hb S loses its fluidity. Binary mixtures of Hb S with Hb A, and with mutant hemoglobins, have been characterized by their "lowest gelling point" as well. More recent experiments, using somewhat different techniques, have evaluated the effect of a diverse population of hemoglobins, both liganded and unliganded, on what has been termed minimum gelling concentration (6). Gelation of deoxy-Hb S has also been studied by sedimentation equilibrium experiments using either interference (7) or schlie...

Paracrystalline fibers of deoxygenated sickle hemoglobin in erythrocytes or concentrated solutions exhibit a phase transformation to a fully crystalline state. X-ray diffraction patterns of the fiber and crystallites are similar except in two respects: the equatorial spacings of the fibers suggest that they pack into a square lattice with a = 220 A, whereas those of the crystals can be indexed on the basis of a net of 187 A by 54 A, and the second-order near-meridional reflections are strong on the fiber pattern but weak on that of the crystallites. The crystallites are isomorphous with single crystals gown in polyethylene glycol solution at pH 4.5 whose structure has been determined at near-atomic resolution (Wishner, B. C., Ward, K. B., Lattmen, E. E. & Love, W. E. (1975) J. Mol. Biol. 98, 179-194). Double filaments of molecules with an axial repeat of 64 A comprise the basic unit of both the crystal and fiber structures. Each filament of the pair is translated with respect to its neighbor by half a molecular diameter along the fiber axis. The two filaments are held together by contacts made by Val 6ft in the molecules of one strand with hydrophobic side chains of the molecule in the neighboring strand. This interaction is probably the cause of the aggregation of filaments into fibers that leads to the sickling of erythrocytes.X-ray diffraction patterns (1) of polymers found in sickled erythrocytes of individuals homozygous for sickle cell hemoglobin (Hb S) and in concentrated solutions of deoxygenated Hb S show a series of sharp meridional reflections arising from a 64-A repeat along the fiber. Potentially, these diffraction patterns contain information about intermolecular contact regions within the polymer, but, because of the poor resolution inherent in cylindrically averaged fiber diagrams and because insufficient parallel alignment of fibers further decreases the resolution, the surface lattice of the polymer has not as yet been established from these patterns; without it the intermolecular interactions cannot be determined.The packing of molecules into the polymers should be evident from electron micrographs, but these show several polymorphic forms, including a 6-stranded microtubular structure (2) and 14-stranded solid elliptical cylinders (3). Occasional sheetlike structures (4), and six-membered discs (5) have also been observed. Such a wide range of polymorphism is difficult to reconcile with our diffraction patterns, all of which appear similar except for differences in resolution. The patterns most likely arise from a single diffracting system.On re-examination of deoxygenated sickled erythrocytes sealed in capillaries several years ago, we have discovered strongly birefringent ribbonlike structures as well as bundles of needles of about 3-&,m diameter and 1-cm length. These were in equilibrium with a water clear solution, which showed that they could not be fibers because the critical equilibrium concentration of the polymer-monomer system of deoxy-Hb S is about 0.22 g-cm-3 (6) under th...

Fibers composed of molecules of deoxygenated sickle cell hemoglobin are the basic cause of pathology in sickle cell disease. The hemoglobin molecules in these fibers are arranged in double strands that twist around one another with a long axial repeat. These fibrous aggregates exhibit a pattern of polymorphism in which the ratio of their helical pitch to their radius is approximately constant. The observed ratio agrees with an estimate of its value calculated from the geometric properties of helical assemblies and the degree of distortion that a protein-protein interface can undergo. This agreement indicates that the radius of an aggregate is limited by the maximum possible stretching of double strands. The geometric properties limiting the radial extent of sickle hemoglobin fibers are fundamental to all cables of protein filaments and could contribute to the control of diameter in other biological fibers such as collagen or fibrin.

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