Heat-induced aggregation and covalent linkages in beta-casein model systems

Conditions and mechanisms leading to covalent aggregation of beta-casein (b eta CN) studied in protein solutions heat treated in the absence or presenc e of glucose. Under all the tested heating conditions, covalent aggregation of beta CN occurred only in the presence of glucose. Only high-MW aggregat es (much greater than 100 kDa) were detectable by polyacrylamide-gel electr ophoresis in the presence of SDS, whereas gel permeation chromatography in the presence of urea showed that aggregates with lower MW were formed as we ll. A characteristic unordered structure was observed using transmission el ectron microscopy for the covalent aggregates of beta CN obtained in the pr esence of glucose, in contrast to the mostly spherical ones due to hydropho bic interactions only. In addition, lysinoalanine (LAL), lysylpyrraline (LP A) and pentosidine (PTD), as possible crosslinking molecules in proteins, w ere quantified by HPLC. The highest amounts of LAL (similar to 150 mmol/mol beta CN) were found in the unglycosylated beta CN, suggesting that this mo lecule is mainly involved in intra-molecular reactions. Very small amounts of LPA (< 2 mu mol/mol beta CN) were found in extensively aggregated beta C N. The behaviour of PTD formation followed that of beta CN aggregation but the low values found (few mmol/mol of protein) suggest that it can only pla y a minor role. Although the covalent aggregation of beta CN is advanced Ma illard reaction dependent, molecules other than those considered here are r esponsible for intermolecular crosslinking but their nature is presently un known. (C) 1999 Elsevier Science Ltd. All rights reserved.