AGGREGATION OF BETA-LACTOGLOBULIN STUDIED BY IN-SITU LIGHT-SCATTERING

In situ light scattering, where light scattered from a sample is measu red directly while the sample is heated in the instrument, is presente d as a simple and effective technique for studying the heat-induced ag gregation of beta-lactoglobulin. This technique was shown to be applic able not only to monitoring the initial aggregation steps, but to foll owing the overall aggregation process with time. The experiments gave results similar to measurements carried out after a heat-quench treatm ent, but were more informative. From experiments on a standard NIZO be ta-lactoglobulin sample, a strongly desalted standard NIZO sample, dif ferent genetic variants of beta-lactoglobulin and a mixture of these, we concluded that the standard NIZO sample was suitable for studying h eat-induced aggregation. This sample has been investigated more extens ively. Results with beta-lactoglobulin (10-100 g/l) at 65 degrees C fi tted a kinetic model for the denaturation and aggregation of beta-lact oglobulin. This model, which held for beta-lactoglobulin dissolved in water at near neutral pH and at 60-75 degrees C. recognizes an initiat ion, propagation and termination reaction, by analogy with polymer rad ical chemistry. It gave a quantitatively correct description of the de pendence of the scattering intensity on the initial beta-lactoglobulin concentration. Salt composition, pH and temperature strongly influenc ed the aggregation of beta-lactoglobulin, Particle size increased with salt concentration in the range studied (up to 20 mM-NaCl and 1.0 mM- CaCl2). When the pH increased from 6.9 to 8.0 particle size was strong ly reduced, whereas it strongly increased when pH was lowered to 6.2. Between 61.5 and 70 degrees C temperature did not affect particle size , whereas aggregation rate strongly increased. These effects could be incorporated in the kinetic model via the reaction constants of the re action kinetic pathway.