Light scattering studies of hemoglobin association

Abnormal protein aggregation has been suggested to be associated with a growing number of diseases. A better understanding of the biophysics and biochemistry of protein aggregation process will be essential to understanding the role of such aggregates in diseases, and critical to preventing or slowing down the aggregation process. The intensity of scattered light from protein solutions is dominated by the concentration fluctuations of the solution. This intensity will increase while aggregates are forming in the solution. We have used light scattering to probe the initial stages of sickle hemoglobin assembly. We employed a novel micro-method for measuring light scattering in a rectangular glass capillary tube that is filled with 24 μL of hemoglobin solution. The solution was illuminated by a laser coming out of an optical fiber that is sealed into the glass tube. The scattered light was collected by a microscope objective at 90° to the incident light, and detected via Photomultiplier tube. The temperature is controlled by a thermoelectric stage. We studied the scattered intensities of five hemoglobin derivatives solution with various concentrations. We found that the intensity of scattered light kept relatively constant at low temperatures but increased after the temperature of the solution exceeded some particular value. Deoxygenated sickle hemoglobin, which forms polymers above solubility, scatters more light than HbSCO (which does not form polymers and differs from deoxygenated HbS by quaternary structural differences), HbACO, deoxygenated HbA, and deoxygenated cross-linked HbA (which do not form polymers and differ from deoxygenated HbS by an amino acid). We interpreted this by using two different theories, liquid-liquid demixing and the appearance of oligomers. We found that monomer and some large oligomer coexisting in the solution, which implies that there might be metastable states in the free energy landscape of hemoglobin aggregation. We obtained the enthalpy changes and entropy changes in the formation of oligomers and found that the enthalpy changes do not show concentration dependence. The axial contact and the lateral contact are found to have different strengths. We found that the data of HbSCO shows surprisingly similar behavior to the data of HbA rather than to that of deoxygenated HbS, which suggest that there are no lateral contacts between the HbSCO monomers. Aggregates concentration is not a diagnostic for polymerization because of that although deoxygenated HbS creates more aggregates at the same concentration and temperature than other hemoglobin derivatives, all derivatives can create equal concentration of aggregates with varied temperature and monomer concentration.Ph.D., Physics -- Drexel University, 201