A B S T R A C T The viscosity of oxygenated blood from patients with sickle cell anemia (Hb SS disease) was found to be abnormally increased, a property which contrasts with the well recognized viscous aberration produced by deoxygenation of Hb SS blood. Experiments designed to explain this finding led to considerations of deformation and aggregation, primary determinants of the rheologic behavior of erythrocytes as they traverse the microcirculation. Deformability of erythrocytes is in turn dependent upon internal viscosity (i.e. the state and concentration of hemoglobin in solution) and membrane flexibility. Definition of the contribution made by each of these properties to the abnormal viscosity of oxygenated Hb SS blood was made possible by analysis of viscosity measurements, made over a wide range of shear rates and cell concentrations, on Hb SS erythrocytes and normal erythrocytes suspended in Ringer's solution (where aggregation does not occur) and in plasma. Similar

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Deoxyhemoglobin from patients homozygous for sickle-cell anemia (deoxyhb S) aggregates into long straight fibers. These may extend through most of the length of the sickled cell, forming either square or hexagonally packed bundles with lattice constants of 170-180 A. Each fiber is a tube made up of six thin filaments, which are wound around the tubular surface with a helical pitch of about 3000 A. Each filament is a string of single hemoglobin molecules linked end to end at intervals of 62 A in dry and 64 A in wet fibers. Molecules in neighboring filaments are in longitudinal register so that they form flat hexagonal rings; these rings are stacked so that successive ones are rotated about the fiber axis by 7.3°. The whole structure repeats after about eight rings. In this structure each molecule makes contact with four neighbors. The likely orientation of the molecules and points of contact between them are discussed. Similar filaments are also observed in normal deoxygenated erythrocytes, but in much lower concentration and aggregated into fibers of irregular diameter. No filaments appear in oxygenated sickle, or normal, adult cells, nor in oxygenated or deoxygenated fetal cells.Sickle-cell anemia is due to a mutation in the globin gene that causes the replacement of one pair of amino-acid residues in the # chains [Glu A3(6),0 Val]. This replacement leaves the solubility of the abnormal hemoglobin in its oxygenated form (oxyhb S) unchanged, but drastically reduces the solubility of deoxyhb S so that it precipitates in the erythrocytes, causing them to become elongated and rigid. The deformation and stiffness of the erythrocytes is the primary cause of the disease and is the feature that distinguishes sicklecell anemia from the anemias caused by other abnormal hemoglobins. If a method could be found of preventing the precipitation of deoxyhb 5, it might lead to a possible therapy.We are approaching this problem by a structural study of the deoxyhb S precipitate.In sickled cells and in cell-free solutions deoxyhb S has been reported to aggregate into fibers with diameters of between 140 and 170 A (1)(2)(3)(4)(5)20). Oriented preparations give x-ray fiber diagrams with a marked periodicity of 64 A along the fiber axis (6). We wish to report electron microscope studies of thin sections of sickled cells and of deoxyhb S that show the general arrangement of the fibers. These are followed by studies of negatively stained preparations in which the arrangement of the individual hemoglobin molecules is resolved, and by further x-ray diffraction work.Several features of the x-ray fiber diagrams can be explained from our findings, but the stereochemical mechanism of aggregation remains to be discovered. METHODSFor electron microscopy of thin sections, deoxygenated sickled cells and ultracentrifuge pellets of deoxyhb S were prepared as described in refs. 3 and 5.Negatively stained specimens of deoxyhb S were obtained by deoxygenating washed erythrocytes from a sickle-cell homozygote suspended in 0.1 M NaCl-1 mM 2,3-...

Direct analyses of solid phase formed by deoxygenating solutions of sickle-cell hemoglobin (Hb S) in the presence of certain other hemoglobin species show that hemoglobins A and C can participate in the filamentous fine structure characteristic of the sickling phenomenon. In contrast, fetal hemoglobin (Hb F) is nearly completely excluded.

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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...

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