Effect of Heat Treatment on the Conformation and Aggregation of β-Lactoglobulin A, B, and C

Abstract

Solutions of bovine β-lactoglobulin (β-Lg) A−C were heated at temperatures between 50 and 90 °C for 12.5 min at pH 6.7 or 7.4, and the products were analyzed by alkaline, sodium dodecyl sulfate (SDS), and two-dimensional (2D) (alkaline and then SDS) polyacrylamide gel electrophoresis (PAGE). Results from the pH 6.7 samples that were ∼70% denatured showed that the proportion of β-Lg that was in very large aggregates was β-Lg C > β-Lg B > β-Lg A. 2D PAGE showed that there were a large number of unexpected intermediate products, especially from β-Lg A. These and other results, including the dissociation of disulfide-bonded dimers from trimers and tetramers by SDS, indicate that (1) β-Lg dimers could be important intermediates in the further aggregation of β-Lg, (2) hydrophobically driven associations occur within the aggregates, (3) the mechanism of β-Lg aggregation is not simple, and (4) differences in variant protein behavior are explainable in terms of net negative charge and specific amino acid substitutions. Keywords: Thermal denaturation; electrophoresis; aggregate formation; hydrophobically associated aggregates; disulfide-linked aggregates; β-lactoglobulin variants; two-dimensional polyacrylamide gel electrophoresis