Faculty Research Interest

Physical Chemistry, Structure, and Pathogenesis of Sickle Cell Hemoglobin

Faculty Record

Sickle cell hemoglobin (HbS) forms long, rod-like, polymers when deoxygenated. These rigidify and distort red cells, producing microvascular obstruction and pathogenesis in sickle cell disease. The HbS system has structural, thermodynamic and kinetic properties in common with other long protein polymers (e.g. tubulin, flagellin, actin, tobacco mosaic virus, beta-amyloid) including helical structure, nucleation controlled kinetics, entropy and excluded volume driven reactions, and phase separation. Thus, it is interesting both for its pathogenic significance and its mechanisms.

HbS polymerization and gelation (i.e. polymer cross-linking) have been studied by physical chemical methods (e.g. light scattering, ultracentrifugation, viscometry, NMR), but these methods reflect average properties of vast numbers of fibers without observation of individual fibers as they nucleate, grow and cross-link. Electron microscopy damages gel structure and fails to reveal events in real time. Conventional microscopy does not resolve the thin (200A diameter) fibers. Using differential interference contrast (DIC) microscopy we overcome these limitations and observe individual HbS fibers in real time as they nucleate, grow and cross-link to form the final solid-like gel (Samuel et al, 1990; Briehl & Guzman, 1994; Briehl, 1995). These studies demonstrate two mechanisms of nucleation of new fibers, mechanisms of fiber growth and melting, diverse cross-linked and branching structures and their mechanisms of formation, and show that fibers are fragile and when broken accelerate polymerization.

In a highly cooperative program project of which I am the principal investigator, we are examining structure, kinetics and mechanisms of the polymerization and melting of hemoglobin S gels. Particular projects in my laboratory include: mechanisms of fiber depolymerization; microrheology of fibers including moduli and persistence lengths; the effects of site specific mutations on equilibria, kinetics, structure and mechanisms; delineation of the molecular sites responsible for cross-linking and nucleation; the role of the red cell membrane. The participating laboratories have expertise in structural biology and modeling techniques, recombinant hemoglobins, kinetic methods and mechanisms and associated physical chemical techniques, modern light microscopic methods, and red cell physiology.