Heat-induced aggregation of beta-lactoglobulin: Role of the free thiol group and disulfide bonds

The heat-induced aggregation of bovine beta-lactoglobulin, dispersed in water at neutral pH and in different concentrations (10, 30, or 50 g of dry matter/L), was studied at 65 degrees C, and the results are related to a kinetic model. Native PAGE and SDS-PAGE analysis under nonreducing and reducing conditions showed that on heating disulfide-linked aggregates were formed and that the average size of these aggregates increased with increasing initial beta-lactoglobulin concentration. In the presence of the thiol-blocking agent N-ethylmaleimide (NEM), at a molar ratio of NEM/beta-lactoglobulin monomer of 1, all thiol groups were blocked and no disulfide-linked aggregates were formed, although with native PAGE high molecular mass noncovalently linked aggregates were observed. The formation of these aggregates accelerated with increasing NEM concentration until a molar ratio of NEM/beta-lactoglobulin monomer of 1 was reached. In separate experiments we studied the effect of pH (in the range pH 6.0-8.0) on the aggregation of beta-lactoglobulin and related this to the pH dependent reactivity of the thiol group.