Abstract
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 (20 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. The monomer on-rate is determined to be 82±2 /mM/Sec. The monomer off-rate is 751±79 molecules/sec in agreement with earlier DIC observations of 850±170 molecules/sec. The method predicts a solubility of 16.0±1.1 g/dl in good agreement with 17.2 g/dl from sedimentation methods. The preceding values are for 25°C. Our measurements also rationalize the observed growth rate of the dense mass of fibers that grows more slowly along the channel and which can be visualized directly. Future uses of this method include HbS fiber bending and HbS solution fluctuations.
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