The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ·mole −1 . First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process N → k D where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.Keywords: Irreversible transitions; scan-rate dependence; scanning calorimetry; chromatography; protein denaturation; aggregation; globular proteins; ovalbumin Aggregation of proteins is an important process in many biological systems and industrial processes. In biological systems it is required for the assembly of structures with specific functions such as microtubules, blood clots, and viral coatings. The formation of plaques is also related to aggregation of specific proteins that have somehow been modified. The aggregation of proteins is, in general, triggered by a conformational change of the protein induced by heat, enzymatic cleavage, or other processes that affect the folded structure. After this change of structure a series of reactions takes place that lead to the formation of aggregates. In many cases it is not clear what drives the formation of specific structures in these aggregates or the formation of fibrils (Thirumalai et al. 2003). Here we present a study of the heat-induced aggregation of chicken egg white ovalbumin. Ovalbumin is known to form fibrillar types of aggregates upon aggregation and, at high enough protein concentrations, a gel can be formed (Weijers et al. 2002b). It is our aim to use ovalbumin as a model system to study how fibrillar aggregates can be formed and what conditions affect the properties of these aggregates. The results are relevant both to understanding the biological function of proReprint requests to: Mireille Weijers,

The effect of ovalbumin net charge on aggregate morphology and visual properties was investigated using chromatography, electrophoresis, electron microscopy, and turbidity measurements. A range of differently charged ovalbumin variants (net charge ranging from -1 to -26 at pH 7) was produced using chemical engineering. With increasing net charge, the degree of branching and flexibility of the aggregates decreased. The turbidity of the solutions reflected the aggregate morphology that was observed with transmission electron microscopy. Increasing the stiffness of the aggregates transformed the solutions from turbid to transparent. Artificially shielding the introduced net charge by introducing salt in the solution resulted in an aggregate morphology that was similar to that for low-net-charge variants. The morphology of heat-induced aggregates and the visual appearance of the solutions were significantly affected by net charge. We also found that the morphology of ovalbumin aggregates can be rapidly probed by high-throughput turbidity experiments.

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