ELECTRON MICROSCOPY OF FIBERS AND CRYSTALS OF A DEOXYGENATED PLATINUM DERIVATIVE OF HEMOGLOBIN S11This work has been supported by a grant from the National Institutes of Health (HL 16005). T. E. W. is supported by Public Health Service Grant No. 5 T32 GM 07281 (MSTP). S. L. W. is supported in part by a Young Pulmonary Investigatorship of the National Institutes of Health (HL 19562). M. W. M. is an Established Investigator of the American Heart Association.

White, Shirley; Wellems, Thomas E.; Fuller, Stephen D.; Quinn, J; Makinen, Marvin W.

doi:10.1016/b978-0-12-164350-8.50027-3

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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%

“…5. These have been previously described from this laboratory (11), and a detailed analysis of the low-order reflections of their optical transforms has been made by Wellems and Josephs (13). A recent analysis of the x-ray diffraction patterns of crystals from stirred solutions free of inositol hexaphosphate has indicated that the crystals are twinned and belong to the triclinic space group P1.…”

Section: Resultsmentioning

confidence: 99%

“…After 2 hr the solutions were transferred to a nitrogen box at ambient room temperature and a few grains of solid sodium dithionite were added to ensure reduction of methemoglobin S and residual oxyHb S. Crystal formation was followed in these vials, and stirring at rates of approximately 1 Hz was continued until crystallization was complete. Aliquots of ;5 ,gl were taken at periodic intervals and were prepared for electron microscopy by described methods (11). Electron micrographs were taken on a Philips EM 300 electron microscope.…”

Section: Methodsmentioning

confidence: 99%

“…Oxygenated Hb S (oxyHb S) was prepared from the blood of individuals homozygous for sickle cell disease as described (11) and was stored in 50% (vol/vol) glycerol at -20'C. Vacuum dialysis against 10 mM phosphate buffer at pH 6.7 containing 0.2 mM EDTA and 10 mM inositol hexaphosphate removed glycerol and concentrated the hemoglobin for crystal preparations.…”

Section: Methodsmentioning

confidence: 99%

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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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