Molecular Modifications of β-Lactoglobulin upon Exposure to High Pressure

Irreversible modifications in tertiary structure, surface hydrophobicity, and association state of β-lactoglobulin were studied after exposure to high pressure (600 and 900 MPa) of solutions of the protein at neutral pH and at different concentrations. Only minor irreversible structural modifications were evident even for treatments as intense as 15 min at 900 MPa. The occurrence of irreversible modifications was time-progressive at 600 MPa but was complete within 2 min at 900 MPa. The irreversibly modified protein was soluble, but some covalent aggregates were formed. Formation of aggregates increased with increasing protein concentration and was prevented by blocking the free thiol moiety in each β-lactoglobulin monomer. Results are discussed in light of their practical relevance, and a unifying denaturation mechanism is envisaged for β-lactoglobulin. In the proposed mechanism, release of monomers represents one of the earliest events, while association of transiently modified monomers stabilizes the denatured forms of the protein.