S. Funtenberger et al., HIGH-PRESSURE PROMOTES BETA-LACTOGLOBULIN AGGREGATION THROUGH SH S-S INTERCHANGE REACTIONS/, Journal of agricultural and food chemistry, 45(3), 1997, pp. 912-921
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-me
rcaptoethanol (MSH), cysteine (CYS), or glutathione (GSH) and then pre
ssurized 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 el
ectrophoresis (PAGE) and PAGE carried out in the presence of sodium do
decyl sulfate (SDS-PAGE) revealed a progressive decrease in beta-Lg fr
om 5 to 30 min, with a corresponding formation of oligomers and high m
olecular weight aggregates, whether pressure was applied in N-2, air,
or O-2. SDS-PAGE with or without MSH demonstrated the progressive incr
ease in S-S-bonded oligomers and aggregates from 5 to 30 min. High con
centrations 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) concentrati
ons of CYS or GSH prevented the formation of S-S-bonded aggregates, pr
obably through interchange reactions between CYS or GSH and the intram
olecular S-S bonds of beta-Lg. These data confirm that most pressure-i
nduced S-S bonds resulted from SH/S-S interchange reactions rather tha
n from oxidation of SH groups.