Light scattering study on the polymerization and aggregation of hemoglobin in high concentration phosphate buffer

Aggregation is a common biological phenomenon that occurs both as a necessary part of normal function as well as in diseased states. It is often an unwanted consequence of misfolded natural and synthetic proteins in applications of biotechnology. Sickle cell hemoglobin (HbS), a mutant form of normal adult hemoglobin (HbA), aggregates into polymers under hypoxic conditions. This polymerization process can be facilitated by the buffer that contains high concentration phosphate.; An understanding of aggregation mechanisms and polymer structure is very helpful for developing treatments for diseases that involve pathological aggregation. Light scattering techniques are useful for studying biological aggregation. Light scattering from a solution of biopolymers can provide information such as the particle size, concentration, shape, and molecular weight. We applied Static Light Scattering (SLS), Dynamic Light Scattering (DLS), and Polarized Light Scattering (PLS) to the study of aggregation and polymerization of both normal and sickle hemoglobin in high concentration phosphate buffer. Using SLS and DLS, we found that aggregates of hemoglobin are formed in high concentration phosphate buffer, which is different from what is found under physiological conditions. We also developed a polar nephelometer to measure PLS from scattering samples. The coupled-dipole approximation, a numerical method for PLS computations, was applied with a parallel algorithm to improve its ability to calculate PLS from large and structurally complex particles.; In this dissertation, we refer to the term of polymerization as a process that is reversible and has product polymers with well-defined structures. The process of aggregation includes, but is not limited to, polymerization. It may be an irreversible process and the product aggregates may be amorphous.