TY - JOUR
T1 - Micromechanics of isolated sickle cell hemoglobin fibers
T2 - Bending moduli and persistence lengths
AU - Wang, Jiang Cheng
AU - Turner, Matthew S.
AU - Agarwal, Gunjan
AU - Kwong, Suzanna
AU - Josephs, Robert
AU - Ferrone, Frank A.
AU - Briehl, Robin W.
N1 - Funding Information:
This work was supported by National Institutes of Health (NHLBI) program project grant HL 58512 (R.W.B. (PI), F.A.F. and R.J.) and grant HL22654 (R.J.). M.S.T. gratefully acknowledges support from The Royal Society (UK) in the form a University Research Fellowship and the extended hospitality of the Center for Studies in Physics and Biology, Rockefeller University.
PY - 2002
Y1 - 2002
N2 - Pathogenesis in sickle cell disease depends on polymerization of deoxy-hemoglobin S into rod-like fibers, forming gels that rigidify red cells and obstruct the systemic microvasculature. Fiber structure, polymerization kinetics and equilibria are well characterized and intimately related to pathogenesis. However, data on gel rheology, the immediate cause of obstruction, are limited, and models for structure and rheology are lacking. The basis of gel rheology, micromechanics of individual fibers, has never been examined. Here, we isolate fibers by selective depolymerization of gels produced under photolytic deliganding of CO hemoglobin S. Using differential interference contrast (DIC) microscopy, we measure spontaneous, thermal fluctuations in fiber shape to obtain bending moduli (κ) and persistence lengths (λp). Some fibers being too stiff to decompose shape accurately into Fourier modes, we measure deviations of fiber midpoints from mean positions. Serial deviations, sufficiently separated to be independent, exhibit Gaussian distributions and provide mean-squared fluctuation amplitudes from which κ and λp can be calculated. λp ranges from 0.24 to 13 mm for the most flexible and stiffest fibers, respectively. This large range reflects formation of fiber bundles. If the most flexible are single fibers, then λp = 13 mm represents a bundle of seven single fibers. Preliminary data on the bending variations of frozen, hydrated single fibers of HbS obtained by electron microscopy indicate that the value 0.24 mm is consistent with the persistence length of single fibers. Young's modulus is 0.10 GPa, less than for structural proteins but much larger than for extensible proteins. We consider how these results, used with models for cross-linking, may apply to macroscopic rheology of hemoglobin S gels. This new technique, combining isolation of hemoglobin S fibers and measurement of micromechanical properties based on thermal fluctuations and midpoint deviations, can be used to study fibers of mutants, hemoglobin A/S, and mixtures and hybrids of hemoglobin S.
AB - Pathogenesis in sickle cell disease depends on polymerization of deoxy-hemoglobin S into rod-like fibers, forming gels that rigidify red cells and obstruct the systemic microvasculature. Fiber structure, polymerization kinetics and equilibria are well characterized and intimately related to pathogenesis. However, data on gel rheology, the immediate cause of obstruction, are limited, and models for structure and rheology are lacking. The basis of gel rheology, micromechanics of individual fibers, has never been examined. Here, we isolate fibers by selective depolymerization of gels produced under photolytic deliganding of CO hemoglobin S. Using differential interference contrast (DIC) microscopy, we measure spontaneous, thermal fluctuations in fiber shape to obtain bending moduli (κ) and persistence lengths (λp). Some fibers being too stiff to decompose shape accurately into Fourier modes, we measure deviations of fiber midpoints from mean positions. Serial deviations, sufficiently separated to be independent, exhibit Gaussian distributions and provide mean-squared fluctuation amplitudes from which κ and λp can be calculated. λp ranges from 0.24 to 13 mm for the most flexible and stiffest fibers, respectively. This large range reflects formation of fiber bundles. If the most flexible are single fibers, then λp = 13 mm represents a bundle of seven single fibers. Preliminary data on the bending variations of frozen, hydrated single fibers of HbS obtained by electron microscopy indicate that the value 0.24 mm is consistent with the persistence length of single fibers. Young's modulus is 0.10 GPa, less than for structural proteins but much larger than for extensible proteins. We consider how these results, used with models for cross-linking, may apply to macroscopic rheology of hemoglobin S gels. This new technique, combining isolation of hemoglobin S fibers and measurement of micromechanical properties based on thermal fluctuations and midpoint deviations, can be used to study fibers of mutants, hemoglobin A/S, and mixtures and hybrids of hemoglobin S.
KW - Fibers
KW - Gel
KW - Micromechanics
KW - Persistence length
KW - Sickle hemoglobin
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U2 - 10.1006/jmbi.2001.5130
DO - 10.1006/jmbi.2001.5130
M3 - Article
C2 - 11812133
AN - SCOPUS:0036307581
SN - 0022-2836
VL - 315
SP - 601
EP - 612
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -