Intermolecular Organization of Deoxygenated Sickle Haemoglobin determined by X-ray Diffraction

Magdoff-Fairchild, Beatrice; Swerdlow, Paul; Bertles, John F.

doi:10.1038/239217a0

Cited by 94 publications

(25 citation statements)

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“…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.…”

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confidence: 81%

“…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.…”

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confidence: 99%

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

Ohtsuki

White

Zeitler

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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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...

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confidence: 81%

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confidence: 99%

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

Ohtsuki

White

Zeitler

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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“…This helical aggregate has been described by Josephs, Edelstein, and coworkers (1, 2, 12, 13) and has been previously observed in slowly stirred solutions (11)(12)(13) This aggregate form of Hb S ranged from a loosely organized assembly of four to five individual fibers to bundles consisting of [12][13][14][15][16][17][18][19][20] individual fibers (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Electron microscope study of the kinetics of the fiber-to-crystal transition of sickle cell hemoglobin.

Wilson¹,

Makinen²

1980

Proc. Natl. Acad. Sci. U.S.A.

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The intermediates and the rate-limiting step in the crystallization of deoxygenated sickle hemoglobin have been determined by a kinetic study with the use of electron microscopy. In slowly stirred solutions of deoxygenated hemoglobin S [Pumphrey, J. & Steinhardt, J. (1977)J. MoL. Biol. 112, 359-3751, the sequential appearance of fibers having a diameter of ;;210 A, bundles of aligned fibers in well-ordered arrays, "thick" fibers of -47O A diameter, and microcrystals is observed. Only the fibers having a diameter of --210 A and bundles of aligned fibers are assigned as kinetically important intermediates of the fiber-to-crystal transition. Addition of microscopic seed crystals obtained from slowly stirred solutions of deoxyhemoglobin S to a solution composed of only fibers and hemoglobin monomers results in more rapid crystallization than in control solutions. Addition of seed crystals after the formation of bundles of aligned fibers does not alter the overall kinetics of crystallization. The results demonstrate that alignment of fibers is the rate-limiting step in the crystallization process and results in formation of nucleation sites for crystal growth.The aggregation of hemoglobin S (Hb S) in deoxygenated solutions is accompanied by changes in several physical properties of the solution. The most important with respect to physiological consequences is the large increase in viscosity attendant with gelation and associated with the formation of helical fibers (1-4). Because the kinetics of gelation exhibit a concentration dependence similar to that observed for condensation processes, the phase transition to a gel has been assumed to require the crystallization of fibers by their alignment (5-8). However, conditions of gentle agitation or stirring inhibit gelation and promote the crystallization of Hb S (9). These observations imply that two physically distinct processes govern the aggregation of Hb S. Because the kinetics of gelation may be a major determinant of the clinical severity of sickle cell anemia (8,10), it is important to define the molecular packing of Hb S in gels and crystals and to characterize those factors that govern its aggregation and polymerization tendencies.In this communication we report the results of an electron microscopic study of the kinetics of crystallization of Hb S in gently agitated solutions. The results demonstrate that fiber formation is part of this crystallization process and that the rate-limiting step in crystal formation is the alignment of helical fibers. Designation of the rate-limiting step in crystal formation as the alignment of fibers requires that gelation is governed by a different rate-determining step and results simply in the entanglement of fibers in highly viscous media. Differentiation of the two rate-governing processes, when correlated with the results of other structural and physiological studies, suggests that gelation and fiber alignment have different roles in the pathophysiology of sickle cell anemia. EXPERIMENTAL PROCEDURESOxygenated H...

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“…This shape change results from aggregation of the sickle hemoglobin (HbS) molecules into parallel filaments [1][2][3][4][5]. Studies of this protein aggregation process have provided much useful information about the molecular mechanism of sickling [1,2,4,[6][7][8][9]. However, the red cell also contains 80% water and one might anticipate that an understanding of the sickling process would be facilitated by a knowledge of the role of water.…”

Section: Introductionmentioning

confidence: 99%