Sickle Hemoglobin Fiber Growth Rates Deduced Using Optical Channels

Abstract

Sickle hemoglobin (HbS) is a point mutant of normal HbA, and will polymerize at concentrations above a well defined solubility. Polymerization occurs by a double nucleation mechanism in which homogeneous nuclei form in solution following a stochastic delay, and heterogeneous nuclei form on other polymers nucleated by either pathway. A fundamental element is the growth of individual fibers, whose diameter (20 nm) precludes direct optical visualization. Fiber growth and depolymerization have been measured by DIC microscopy, but the heterogeneous pathway makes it possible that bundles rather than single fibers have been observed, in addition to certain technical problems that make interpretation of the results less than simple. We have devised a method in which optical patterns are projected on a COHbS solution by laser photolyis, which creates deoxyHbS that can polymerize only in the illuminated area, easily allowing complex polymer structures to be created optically. In our experiment, polymers first form in an incubation circle. From the circle, a line of deoxyHbS is optically generated along which fibers can grow. Finally, a detection circle is illuminated and the connecting line is extinguished. If a polymer has entered the detection circle, thanks to heterogeneous nucleation it will fill the circle with easily observed polymers, otherwise the detection circle remains monomeric until, at some much later time, homogeneous nucleation occurs. Thus we can measure the elongation of a fiber too small to detect optically. Our preliminary results find growth rates greater than previously observed by DIC microscopy. We can also measure the growth rate of bundles by observing a mass of fibers that subsequently grow along the channel, and these grow at rates comparable to those determined by DIC microscopy. Implications of the measurements and the method will be discussed.