High Pressure Promotes β-Lactoglobulin Aggregation through SH/S−S Interchange Reactions

Abstract

Solutions of β-lactoglobulin (β-Lg) isolate (23 g of protein/kg, pH 7.0, in 50 mM Bis-Tris buffer) were either flushed with N2 or O2 or brought to given concentrations of N-ethylmaleimide (NEM), β-mercaptoethanol (MSH), cysteine (CYS), or glutathione (GSH) and then pressurized at 450 MPa and 25 °C for 5, 15, or 30 min. Sulfhydryl groups (SH), half-cystine residues, and S−S bonds were not influenced by pressure (0−30 min), with or without prior flushing with N2, thus revealing no significant oxidation of SH groups. Polyacrylamide gel electrophoresis (PAGE) and PAGE carried out in the presence of sodium dodecyl sulfate (SDS−PAGE) revealed a progressive decrease in β-Lg from 5 to 30 min, with a corresponding formation of oligomers and high molecular weight aggregates, whether pressure was applied in N2, air, or O2. SDS−PAGE with or without MSH demonstrated the progressive increase in S−S-bonded oligomers and aggregates from 5 to 30 min. High concentrations of NEM (30× the SH group content, on a molar basis) or of MSH (50×) prevented the pressure-induced formation of all aggregates, or only of S−S-bonded aggregates, respectively. High (30×) concentrations of CYS or GSH prevented the formation of S−S-bonded aggregates, probably through interchange reactions between CYS or GSH and the intramolecular S−S bonds of β-Lg. These data confirm that most pressure-induced S−S bonds resulted from SH/S−S interchange reactions rather than from oxidation of SH groups. Keywords: Hydrostatic pressure; β-lactoglobulin; aggregation; disulfide bonds; sulfhydryl groups; SH/S−S interchange reactions; intermolecular S−S