Thiol Reactivity in Pressure-Unfolded beta-Lactoglobulin. Antioxidative Properties and Thermal Refolding

Pressure treatment of beta-lactoglobulin (0.11 mM in aqueous 0.16 M NaCl, pH 7.61, at 15 degrees C for 30 min, up to 400 MPa investigated) induces antioxidative properties as shown for linoleic acid peroxidation in oil-in-water emulsions. The antioxidative properties obtained through pressure treatment are gradually lost at ambient pressure and paralleled by a decrease in thiol exposure and reactivity, as determined with Ellman's reagent, in an entropy-controlled (DeltaS() = -247 +/- 7 J mol(-)(1) K(-)(1)) first-order renaturation process (half-life of 3.1 h at 25 degrees C, pH 7.61, independent of pressure used for denaturation at least up to 250 MPa) with a modest temperature dependence (DeltaH() = 23 +/- 2 kJ mol(-)(1)). The reactivity of the thiol group toward Ellman's reagent was studied kinetically by stopped-flow spectrometry. The apparent second-order rate constant for this reaction at pH 7.61 and 25 degrees C changes from 5.7 x 10(2) L mol(-)(1) s(-)(1) for native beta-lactoglobulin to 1.6 x 10(5) L mol(-)(1) s(-)(1) for beta-lactoglobulin pressure-denatured at 200 MPa. Half-denaturation occurred at approximately 50 MPa. The degree of exposure of the thiol group corresponds to half-denaturation around approximately 140 MPa with a reaction volume, DeltaV degrees, for denaturation of -61 +/- 3 mL.mol(-)(1), a difference in half-denaturation pressure which may indicate that pressure denaturation is a stepwise process.