Pressure-induced denaturation of monomer β -Lactoglobulin-B is partially irreversible

摘要

This experiment was conducted to assess the effect of high hydrostatic pressure on monomer β -Lactoglobulin-B (BLg) at acidic pH by fluorescence spectroscopy under pressure and by circular dichroism and ~1H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of the center of spectral mass or ANS fluorescence was almost complete upon the pressure release. No difference in the ~1H NMR spectrum was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of amide protons indicated that the conformation at the vicinity of tryptophan residues can be refolded almost completely after release of pressure. These results confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost although its retinol binding ability was the same as in the unpressurized species. Furthermore, the CD spectra of the far-UV region and the 2D ~1H NMR spectra clearly revealed the difference in the molecular conformation between unpressurized and pressurized BLg. These results are interpreted in terms of the existence of a partially fragile structure in the BLg molecule due to high pressure.