Fundamental studies of sickle hemoglobin polymerization

Zenghui Liu

doi:10.17918/etd-3439

The key to the pathology of sickle cell disease is the polymerization of sickle hemoglobin (HbS), which is a point mutation of the normal hemoglobin (HbA). Thepolymerization of HbS occurs when the concentration of deoxy HbS exceeds the solubility. If inert molecules such as dextran are introduced to the solution, thesolubility could be reduced significantly because of crowding. This makes the study of the solubility with limited amount of sample possible. By applying scaled particle theory, we have built the thermodynamic connection between the situation with and without dextran, which enable one to calculate the dextran-free solubility easily. The HbS polymerization could be modeled by the double nucleation mechanism. However, a fundamental element of this mechanism, the growth speed of individualpolymer is still not precisely measured because the single polymer is 20nm diameter, thus below optical resolution. Our approach is based on the fact that a single fiber entering a region of concentrated deoxy HbS will generate large numbers of additional polymers by heterogeneous nucleation, allowing the presence of the firstfiber to be inferred even if it is not directly observed directly. This idea is realized by projecting an optical pattern consisting of three parts: a large incubation circle, a small detection area, and a thin channel connecting the two areas to the sample. With increasing the channel on time, we can find the time just enough for the polymerreach the detection circle, and with the channel length measured, the polymer growth speed could be calculated. Our polymer growth rates obtained from pure HbS,HbS/HbA mixtures, and partial photolysis of HbS validate a simple linear growth rate equation including any non-polymerizing species in the activity coefficientcalculation. This implies that monomer is adding to the end of the polymer one by one not by oligomers. Our approach also enables us to determine the monomer addition rates and release rates precisely, and their temperature dependence. These data are in agreement with previous DIC measurement. The ratio of these two rates is the solubility of individual polymer, which also agrees with the previously published centrifugation data.

Fundamental studies of sickle hemoglobin polymerization

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Fundamental studies of sickle hemoglobin polymerization

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