Characterization of intermediates formed during heat-induced aggregation of beta-lactoglobulin AB at neutral pH

摘要

Heat-induced aggregation of beta-lactoglobulin AB (17 mg/ml in 0.005 M NaCl; pH 7.0; 78.5oC) was studied using size-exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. By choosing the chromatographic condition such that the native beta-lactoglobulin was partially associated, the formation of irreversibly altered monomers and non-native dimers could be monitored. Heat-induced aggregation of beta-lactoglobulin occurred via many intermediates, and mainly via disulfide bonding and to a much lesser extent via non-covalent interactions. In the early stages of aggregation of beta-lactoglobulin, non-native dimers and oligomers were formed, presumably mainly via thiol-disulfide exchange and to a lesser extent via thiol-thiol oxidation and non-covalent interactions. In later stages, aggregates with a continuous distribution of molar masses from 105 to 2X106 Da were formed, presumably via the incorporation of monomers and small aggregates into larger aggregates mainly through disulfide bonding.