Kinetics of domain formation by sickle hemoglobin polymers

Abstract

We have monitored the growth of domains of sickle hemoglobin polymers by using temporally and spatially resolved light scattering and birefringence measured pseudosimultaneously on a 50-microns square area. Polymerization was induced and indefinitely maintained by photolysis of the carbonmonoxy derivative using an argon ion laser. Intensity of scattering and birefringence (measured as intensity transmitted through crossed polarizers) were measured using a silicon-intensified target vidicon interfaced to a computer. Polymer concentration, as inferred by light scattering, grew with primarily circular symmetry, with approximately 20% of the signal initially in a twofold symmetric pattern. In time the circular symmetry increased. A distinct decrease in the scattering signal developed which spread outward from the center of the domain. Birefringence lagged the scattering and initially grew in a twofold pattern, with the formation of a characteristic Maltese cross only appearing much later, and well after the scattering signal had peaked. Radial profiles of the domain scattering and birefringence were both approximately gaussian. We successfully modeled the decrease in scattering by fitting the profiles to a large gaussian from which a second smaller gaussian was subtracted. This second gaussian had the width of the birefringence gaussian. The width of the birefringence gaussian grew linearly in time, while the width of the scattering gaussian showed a notable acceleration. We conclude that domains form primarily as disordered arrays which align at later times. We explain the above observations, including the shape of the birefringence progress curves, as the result of an alignment transition which is solely due to a redistribution of monomers from short to long, and from entangled to radial, polymers. We present a theoretical justification for this process in an appendix. In a separate paper (Zhou, H. X., and F. A. Ferrone, manuscript submitted for publication) we show that the gaussian shapes and acceleration of the width naturally arise from a generalization of the double nucleation mechanism for sickle hemoglobin gelation (Ferrone, F. A., J. Hofrichter, H. Sunshine, and W. A. Eaton 1980. Biophys. J. 32:361-377; Ferrone, F. A., J. Hofrichter, and W. A. Eaton. 1985. J. Mol. Biol. 183:611-631).