PP3 - Role of oxidation and thermal unfolding in structural changes to beta-lactoglobulin

Abstract

Oxidation is a major cause of protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, with this giving rise to changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, susceptibility to digestion and function. Whilst it is well established that these processes have negative impacts on product quality and human health, the mechanisms that generate these alterations are incompletely understood. In this project we are examining whether and how unfolding and oxidation induce changes in the structure of beta-lactoglobulin, to determine whether unfolding alters the rate and mechanism by which samples undergo oxidation, and vice versa. Gaining an understanding of how this occurs is critical to the development of methods to stop undesirable changes, particularly in dairy products where heat treatment is widely used, and oxidation is often encountered. Heat treatment has been shown to result in unfolding of beta-lactoglobulin as assessed by circular dichroism measurements. This effect was increased, in a dose-dependent manner, in the presence of added H 2 O 2 . Addition of high doses of H 2 O 2 before heating also resulted in a shift of the intrinsic (Trp-derived) protein fluorescence maximum to higher wavelengths. Thiol quantification, using 5,5’-dithio-(2-nitrobenzoic acid), revealed that H 2 O 2 depletes thiols in a dose-dependent manner. Using SDS-PAGE, it was shown that whilst heating induced reducible cross-links; pretreatment with high levels of H 2 O 2 prevented the formation of these species. With lower concentrations of H 2 O 2 , reducible cross-links were generated during subsequent heating, consistent with a key role for disulphide formation in heat-induced cross-link formation, with H 2 O 2 diminishing aggregate formation. Altogether, these data indicate that both thermal treatment and oxidation can induce modifications to the protein via competing processes, and alter the vulnerability of the protein to aggregation.