Structure of Hemoglobin S Fibers: Optical Determination of the Molecular Orientation in Sickled Erythrocytes

Abstract

Possible orientations of deoxyhemoglobin S molecules within sickle-cell fibers are delimited by polarized absorption measurements on single sickled cells and single crystals of deoxyhemoglobin A. The polarization ratio of cells provides a lower limit for that of an individual fiber and, coupled with the absorption properties of the deoxyhemoglobin molecule, restricts the orientation of the long molecular (x) axis to within 220 of the fiber axis. Adopting the stacked ring model of Finch et al. for the molecular positions and the additional constraint that at least one mutated (G6) site is part of an intermolecular contact, our optical result requires that the true molecular dyad (y) axis pass through some part of an adjacent molecule in the same ring. This range of orientations for the y axis is approximately perpendicular to those described in existing models and places at least one 36 residue in position to be part of a contact between molecules in the same ring.The deformation that accompanies the deoxygenation of erythrocytes from patients with sickle-cell anemia is believed to result from the aggregation of hemoglobin molecules into an ordered phase. Crystallinity is evident from the linear birefringence (1-6) and dichroism (7,8), and from the fibertype x-ray diffraction patterns (9) of sickled cells and gels of deoxyhemoglobin S. Electron-microscope studies (6,8,(10)(11)(12)(13)(14) indicate that the ordered phase is composed of bundles of long straight fibers, aligned parallel to each other as in a nematic liquid crystal. Finch et al. (8) have proposed a model for the individual fibers ( Fig. 1), which appears to be consistent with both the electron-microscope and x-ray diffraction results. In this model the fiber may be described as a microtubule constructed from stacked rings of six hemoglobin molecules, where each ring is rotated slightly relative to the one below it. The structure can alternatively be viewed as six intertwined helical filaments, each having about 48 hemoglobin molecules per turn. If this model for the molecular positions is correct, then a knowledge of the precise molecular orientation should define the role of the mutation sites [Glu A3(6)0--B Val] in forming intermolecular contacts within the fiber.Neither electron microscopy nor x-ray diffraction has provided any concrete information on the molecular orientation. The hemoglobin molecule is a slightly elongated spheroid of dimensions 65 X 55 X 50 i (15). The lack of significant shape anisotropy permits considerable freedom in packing hemoglobin molecules into a fiber of dimensions compatible with those found by electron microscopy and x-ray diffraction. In contrast to the physical ellipsoid, the ellipsoid that describes the absorption of plane-polarized light by a hemoglobin molecule is highly anisotropic (Fig. 2). One of the dimensions of this ellipsoid (x) is considerably shorter than the other two, and it is possible to obtain its precise orientation within the fiber from polarized absorption measurements. Visual observati...

Kinetics and Mechanism of Deoxyhemoglobin S Gelation: A New Approach to Understanding Sickle Cell Disease

“…The large number of coupled differential equations must, in general, be solved by numerical techniques. For short times, however, the concentration of the aggregate of i monomers, when treated by the above scheme, can be shown by iterative integration to be given by [3] At longer times, additional terms become important, and the rate of appearance decreases, the concentration of i-mer eventually becoming invariant when the system approaches the steady state. This time dependence is very similar to that described in detail by Abraham (25) for the condensation of water from supersaturated air.…”

Electron microscopy of fibers and discs of hemoglobin S having sixfold symmetry

“…The erythrocyte sickling phenomenon of sickle cell anemia is associated with aggregation of hemoglobin S (HbS) molecules into linear arrays or fibers (1)(2)(3)(4)(5)(6)(7)(8)(9). These aggregates form long bundles of fibers within the erythrocyte and are believed to be responsible for deformation of the cell into abnormal shapes with the consequent acute physiologic manifestations known as sickle cell crisis.…”

“…Structural features of aggregates of deoxyhemoglobin S (deoxyHbS) molecules have been characterized by electron microscopy (1-5), x-ray diffraction (6), and physical chemical studies (7)(8)(9)(10). On the basis of these studies, models have been presented that are consistent with the fl6 site at the points of intermolecular contact (12); however, direct structural determination of the molecular orientation of the deoxyHbS molecule in the fiber is required to resolve the role of the substituted valine residue.…”

Effect of Alkylureas on the Polymerization of Hemoglobin S

“…Intracellular ordered aggregates have been demonstrated in deoxygenated erythrocytes from persons with sickle cell anemia and studied by electron microscopy (3)(4)(5)(6), by measurements of low-angle x-ray diffraction (7), linear birefringence (8), and dichroism (6,9). The polymers, elongated microtubules having diameters of about 180 A, deform the erythrocyte (sickling) and alter their rheological properties.…”