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

Abstract

Aggregated forms of deoxyhemoglobin S were examined with a field emission transmission electron microscope. Images of isolated helical fibers were obtained from sickled cell lysates stained directly on the electron microscope grid. Optical and digital analyses of the electron micrographs showed that the fibers are similar to those characterized by J. T. Finch, M. F. Perutz, J. F. Bertles, and J. Dobler [(1973) Proc. NatL Acad. Sci. USA 70, 718-7221 in that they consist of stacked discs each composed of six hemoglobin molecules. The fibers exhibit an outer diameter of 160-170 A and an inner diameter of about 60 A with an axial spacing of 58 A per disc. The fiber can be described as a helix consisting of 56 discs per helical turn. We observed discs of six hemoglobin molecules, which may be stable substructural components of the fibers. They were observed in preparations of hemoglobin fibers and exhibited 6-fold symmetry by power spectrum analysis. A reconstructed image of a disc digitally filtered for 6-fold symmetry has a maximum external diameter of -170 A and a central hole of 60 A diameter and is similar to the axial projection of a single disc from a low-resolution, three-dimensional reconstructed model of a fiber.The erythrocyte sickling phenomenon of sickle cell anemia is associated with aggregation of hemoglobin S (HbS) molecules into linear arrays or fibers (1-9). 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. The molecular basis of sickle cell anemia is a mutation in the 13-globin gene causing replacement of Glu A3(6)# with valine in each of the subunits of hemoglobin (11).It is clearly desirable to relate the structural alteration of the hemoglobin molecule to the mechanism responsible for the formation of the characteristic linear aggregates. 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.Correlating the features of the fibers with the chemical and physical properties of the deoxyHbS molecule has been complicated by the fact that at least two distinct types of fibers have been described by electron microscope studies: a six-stranded helix (1, 2) having 6-fold axial symmetry and two types ofThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. eight-stranded helices in which pairs of st...

Cross-sectional views of hemoglobin S fibers by electron microscopy and computer modeling.

“…Deviations from the z axis are defined by the angle 0, and rotation of the 0-vector about the z axis is defined by the angle k. Small values of 0 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20),can lead to appreciable changes in the appearance of the model cross sections, as indicated in Fig. 3 d-f.…”

“…Studies on the structure of the fibers of Hb S have focused on two forms: a t170-A-diameter form with a 6-strand, stackeddisk structure first described by Finch et al (1) and more recently investigated by Ohtsuki et al (2); and a z200-A-diameter form with a helical structure recently described by Dykes et al (3) in terms of a 14-strand model. A major question has been Which of these forms predominates in sickled cells?…”

Structure of a crystal form of human methemoglobin indicative of fiber formation

“…It has been noted that sickle-cell hemoglobin fibers are frequently polymorphic (Finch et al, 1973;Ohtsuki et al, 1977;Magdoff-Fairchild & Chiu, 1979;Kaperonis et al, 1986;Mu & Magdoff-Fairchild, 1992;Wang et al, 2000), which suggests that the fiber structure may be variable or ill-defined or that it consists of more than one component. There is also evidence that structural transformations may occur in the filaments making up sickle fibers as a consequence of time or other physical factors (Magdoff-Fairchild & Chiu, 1979;Kaperonis et al, 1986;Mu & Magdoff-Fairchild, 1992).…”

Sickle-cell hemoglobin: fall in osmotic pressure upon deoxygenation.

“…Subsequently, comlete gelation of these agegates could cause additional water loss and thrust the sickled cell into an irreversible cycle. The osmotic pressure of normal hemoglobin does not change appreciably during deoxygenation and is essentially the same as the osmotic pressure of oxygenated sickle-cell hemoglobin.Sickling of deoxygenated erythrocytes containing hemoglobin S (Hb S) results from an aggregation of Hb S tetramers into a gel of long, multistranded polymers (1)(2)(3)(4)(5) …”

“…Sickling of deoxygenated erythrocytes containing hemoglobin S (Hb S) results from an aggregation of Hb S tetramers into a gel of long, multistranded polymers (1)(2)(3)(4)(5) …”