Real-Time Monitoring of Heat-Induced Aggregation of beta-Lactoglobulin in Aqueous Solutions Using High-Resolution Ultrasonic Spectroscopy

High-resolution ultrasonic spectroscopy was applied for real-time monitoring of the heat-induced denaturation and aggregation processes in aqueous solutions of beta-lactoglobulin. The temperature profiles for the ultrasonic velocity and attenuation in the frequency range from 4MHz to 16MHz were measured during heating and cooling cycles, 35A degrees C to 120A degrees C to 35A degrees C, with different heating and cooling rates. Two processes were identified in the heating profiles: transition to the molten globular state followed by formation of protein aggregates. Both processes are accompanied by a decrease in the ultrasonic velocity and an increase in compressibility. The ultrasonic attenuation did not show a significant change during the transition to the molten globule but increased significantly during aggregation. The diameter of the aggregates (calculated from ultrasonic attenuation) was of the order of 100nm and depended on the pH and the heating rate. Variation of pH from 6.0 to 7.5 had a pronounced effect on the size of protein aggregates. Some effect of pH on the intrinsic properties of aggregates was also detected.