Frank Anthony Ferrone

Sickle hemoglobin (HbS), a mutant of normal adult hemoglobin (HbA), will polymerize at concentrations above a well-defined solubility. HbS polymerization occurs by a double nucleation mechanism. A fundamental element of the mechanism is the growth of individual fibers, whose diameter (21 nm) precludes direct optical visualization. We have developed a photolytic method to measure the HbS fiber growth speed in HbS carbon monoxide derivative (COHbS) solutions. The idea of this method is that a single fiber entering a region of concentrated deoxyHbS will generate large numbers of additional fibers by heterogeneous nucleation, allowing the presence of the first fiber to be inferred even if it is not directly observed optically. We implement this method by projecting an optical pattern consisting of three parts: a large incubation circle, a small detection area, and a thin channel connecting the two. The connecting channel is turned on for just a short time; only if fiber growth is fast enough will the detection circle polymerize. Our fiber growth rates obtained from pure HbS, HbS/HbA mixtures, and partial photolysis of HbS validate a simple growth rate equation including any non-polymerizing species in the activity coefficient calculation. For 25 degrees C we have determined the monomer on-rate to be 82 +/- 2 mM(-1)s(-1). The monomer off-rate is 751 +/- 79 molecules/sec in agreement with earlier DIC observations of 850 +/- 170 molecules/sec. Combining the above, the method predicts a solubility of 16.0 +/- 1.1 g/dl in good agreement with 17.2 g/dl measured from sedimentation methods.