ß-Lactoglobulin is the main protein in the whey protein fraction of bovine milk. The protein shows a natural reversible non covalent binding ability for small hydrophobic compounds – such as vitamins, fatty acids and polyphenols. Irreversible covalent binding to thiol and amino groups of this protein is also possible with some reactive ligands, such as allyl isothiocyanate (AITC). Non-covalent and covalent modifications of proteins are possible means to incorporate bioactive ligands to food matrices. These interactions can alter the proteins physicochemical abilities – making it more hydrophobic or changing the proteins tertiary structure.
We were able to show with different methods (fluorescence quenching, equilibrium dialysis, headspace-water equilibrium, and mass spectrometry) that 5 molecules of AITC interact covalently with 1 molecule of ß-LG at pH 8.5. This reaction showed positive cooperative ligand binding – i.e. the binding of one molecule of AITC led to changes of the proteins tertiary structure – revealing another binding site with higher affinity than the first binding site. AITC binding had also impact on the tryptic digestion of the protein. Several cleavage sites of the protein for trypsin were blocked by ligand binding, resulting in different peptides. The understanding of ligand binding to proteins gives further insights into directed protein modifications in food matrices.
Julia Katharina Keppler jkeppler@foodtech.uni-kiel.de
