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,

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The effect of ionic strength on the interaction of ovalbumin, a globular egg white protein, in aqueous solution was investigated using static and dynamic light scattering. Strong repulsive interactions are observed at low ionic strength (3 mM). Aggregation of the proteins was induced by heating at low (3 mM) and high (100 mM) ionic strength as a function of the concentration. The size of the aggregates increases with increasing protein concentration and diverges close to the critical concentration for gelation, which is about 60 g/L at low ionic strength and 12 g/L at high ionic strength. Static and dynamic light scattering showed that at low ionic strength linear chains are formed with little branching until close to the gel point, while at high ionic strength denser branched aggregates are formed with a fractal dimension close to that found for other globular protein aggregates. The observations were confirmed by cryo-transmission electron microscopy. Heated systems at low ionic strength remained transparent and were studied in situ using static and dynamic light scattering. The relaxation of the concentration fluctuations occurs by cooperative diffusion, except when the gel point is approached and a slow secondary relaxation process is observed. The slow mode is attributed to the self-diffusion of the aggregates and restructuring of the system. The terminal relaxation time of the concentration fluctuations diverges in the neighborhood of the gel point because a fraction of the concentration fluctuations is progressively frozen in.

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.

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