Thermal stability of [alpha]-lactalbumin

by (Matthew Kenneth), 1975- McGuffey

McGuffey, Matthew Kenneth. Thermal Stability of ?-Lactalbumin. (Under the Direction of E. Allen Foegeding.) The first objective of this research was to quantitatively describe the denaturation and aggregation processes of ?-lactalbumin at neutral pH in order to understand their interrelationship and effect on protein stability. Three different preparations of ?-La (two pure and one commercial) had similar denaturation temperatures, enthalpies and % reversibility values as measured by differential scanning calorimetry. However, heated pure preparation reveled three non-native monomer bands that corresponded to three distinct dimer bands (as measured by Native PAGE). This suggested that specific intramolecular disulfide bond shuffling lead to formation of disulfide-specific dimers. The apo protein was the most thermostable to turbidity development and this was independent of preparation. The C?-La was the most thermostable holo- preparation. Turbidity development at 95°C (?95°C) suggested pure preparations intensely associate through hydrophobic interactions through bridging by divalent phosphate and this effect was mitigated by decreasing the ionic strength, decreasing the phosphate charge to –1 (at pH 6.6) or decreasing the temperature. The second objective was to investigate the aggregation behavior of a commercial ?-La at neutral pH and 95°C in a nutritional beverage mineral salt environment. The variables explored were ?-La lot variation, relative ?-lactoglobulin concentration and excess calcium on the aggregate size development as measured by light scattering and turbidity development. The lot of holo-?-La possessing a higher intrinsic ?-Lg concentration had higher solubility at pH ? 6.80, evolved more reactive thiol groups, had a 25% faster first order rate constant, dissociated only slightly with cooling and formed spherical aggregates with a much higher molecular weight. Aggregates intrinsic to the protein powder may play a role in aggregate growth and shape. Adding increasing quantities of ?-Lg generally decreased solubility. The highest ?-Lg concentrations investigated demonstrated a net thiol oxidation and, subsequently, had a diminished ability to aggregate through hydrophobic interactions. Adding excess calcium caused a dramatic loss of solubility at pH 7.0 and required an increase in pH to 7.5 to regain solubility. Thermal Stability of ?-Lactalbumin by Matthew K. McGuffey A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Food Science Raleigh 2004