Glycation of bovine β-Lactoglobulin: effect on the protein structure |
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Authors: | Franç ois Morgan,Daniel Mollé ,Gwé naë le Henry,Annie Vé nien,Joë lle Lé onil,Gabriel Peltre,Didier Levieux,Jean-Louis Maubois,& Saï d Bouhallab |
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Affiliation: | Institut Technique des Produits Laitiers Caprins, BP-49, F17700 Surgères, France;INRA-LRTL, Biochemistry Unit, 65 rue de Saint Brieuc, F-35042 Rennes cedex, France;INRA-SRV, Immunochemistry Unit, F-63122 Saint-Genès-Champanelle, France;Institut Pasteur, Immunoallergy Unit, 28 rue du Docteur Roux, F-75015 Paris, France |
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Abstract: | Summary Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60 °C) in an aqueous solution and after a treatment under a restricted water environment (50 °C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers 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 β-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. |
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Keywords: | β-Lactoglobulin binding sites conformation denaturation glycation lactose |
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