Glycation of bovine beta-Lactoglobulin: effect on the protein structure

Since temperature and water activity are among the most important parameter s that affect the Maillard reaction, the glycation sites in pure, native bo vine beta-lactoglobulin were determined after a mild heat treatment (60 deg rees C) in an aqueous solution and after a treatment under a restricted wat er environment (50 degrees C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of beta-lactoglobu lin (beta-LG) glycoforms with respect to the number of lactose residues lin ked per protein molecule and to the binding sites involved. Subsequently, t he effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated beta-LG were characterised by p roteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-nap htalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The resu lts showed that dry-way glycation did not significantly alter the native-li ke behaviour of the protein while the treatment in solution led to importan t structural changes. These changes resulted in a specific denatured beta-L G monomer, which covalently associated via the free thiol group. The homodi mers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non-covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native b eta-LG, confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein .