Influence of binding conjugated linoleic acid and myristic acid on the heat- and high-pressure-induced unfolding and aggregation of β-lactoglobulin B

Abstract

Heat and pressure treatment of β-lactoglobulin B (β-LG) causes it to partially unfold and aggregate. β-LG solutions at pH 7.2 were heat treated at temperatures between 40 and 93 °C for 12 min or were pressure treated at pressures between 50 and 800 MPa for 30 min. Another set of samples also contained myristic acid (MA) or conjugated linoleic acid (CLA) in a molar ratio of 1:1.1 protein:ligand. All the treated samples were analysed using polyacrylamide gel electrophoresis (PAGE) and near- and far-UV circular dichroism (CD) spectroscopy. Native- and sodium dodecyl sulfate (SDS)-PAGE showed that, at temperatures above about 63 °C, bands of lower mobility were produced. As the treatment temperature increased, the quantity of native protein decreased steadily and the quantity of higher molecular weight aggregates increased. Thus β-LG could be native (Stage I T) or non-native disulfide-bonded monomers and polymers (and their stable hydrophobic adducts) (Stage II T). Pressure treatments of 50 and 100 MPa had no discernible effect. At pressures between 150 and 350 MPa, PAGE analysis showed that only non-native and dimer β-LG were produced but that hydrophobic adducts were apparent in the native-PAGE patterns. At pressures above 500 MPa, the whole range of polymers (higher molecular weight aggregates) was discernible. Therefore three stages have been proposed for the pressure denaturation of β-LG. Thus β-LG could be native (Stage I P), non-native disulfide-bonded monomers, intermediates in the aggregation process and dimers (and their stable hydrophobic adducts) (Stage II P) or larger polymers (Stage III P). This model is consistent with the near- and far-UV CD data and with reported 8-anilino-1-naphthalenesulfonate probe data. The addition of MA or CLA to β-LG followed by heat or pressure treatment shifted the transition of native β-LG to non-native monomer or dimer by stabilizing the native structure (Stage I T or Stage I P). Pressurizing similar samples at higher pressures showed that only CLA had the ability to inhibit the transition from Stage II P to Stage III P.