Modification of the single unpaired sulfhydryl group of beta-lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation [Single SH of beta-lactoglobulin and pressure denaturation]

Chemical modification reactions of the unpaired sulfhydryl group of beta-lactoglobulin (LG) under high pressure and the role of this group in the pressure-induced denaturation were investigated. When LG was incubated at 400 MPa (pH 6.8) for 1 h, dimerization through intermolecular reaction of SH was observed. The generation of the covalently linked dimers were prevented by the presence of N-ethylmaleimide (NEM), an agent for SH-specific modification. The reactivity of the SH group of LG, which is buried inside in its native state, was increased by high pressure, as a result of its exposure to the protein surface accompanied by the pressure denaturation. The effect of NEM was also observed in the fluorescence change caused by high pressure, in both the intrinsic fluorescence of LG and the retinol fluorescence of the LG-retinol complex. The control showed an irreversible change at neutral pH, but it became mostly reversible in the presence of NEM. Compatible results were obtained by CD spectroscopy. Inter- and intramolecular reactions of the SH group are suggested to be main causes for the pressure-induced irreversible denaturation of LG.