High pressure promotes beta-lactoglobulin aggregation through SH/S-S interchange reactions

Solutions of beta-lactoglobulin (beta-Lg) isolate (23 g of protein/kg, pH 7.0, in 50 mM Bis-Tris buffer) were either flushed with N-2 or O-2 or brought to given concentrations of N-ethylmaleimide (NEM), beta-mercaptoethanol (MSH), cysteine (CYS), or glutathione (GSH) and then pressurized at 450 MPa and 25 degrees 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 N-2, 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 beta-Lg from 5 to 30 min, with a corresponding formation of oligomers and high molecular weight aggregates, whether pressure was applied in N-2, air, or O-2. 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 (30x the SH group content, on a molar basis) or of MSH (50x) prevented the pressure-induced formation of all aggregates, or only of S-S-bonded aggregates, respectively. High (30x) 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 beta-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.