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 Na Cl, pH 7.61, at 15 degrees C for 30 min, up to 400 MPa investigated) i nduces antioxidative properties as shown for linoleic acid peroxidatio n in oil-in-water emulsions. The antioxidative properties obtained thr ough pressure treatment are gradually lost at ambient pressure and par alleled by a decrease in thiol exposure and reactivity, as determined with Ellman's reagent, in an entropy-controlled (Delta S double dagger = -247 +/- 7 J mol(-1) K-1) first-order renaturation process (half-li fe 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 depend ence (Delta H double dagger = 23 +/- 2 kJ mol(-1)). The reactivity of the thiol group toward Ellman's reagent was studied kinetically by sto pped-flow spectrometry. The apparent second-order rate constant for th is 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-denaturat ion occurred at similar to 50 MPa. The degree of exposure of the thiol group corresponds to half-denaturation around similar to 140 MPa with a reaction volume, Delta V 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.