Pressure-induced denaturation of monomer beta-lactoglobulin is partially irreversible: Comparison of monomer form (highly acidic pH) with dimer form (neutral pH)

This study was conducted to assess the effect of high hydrostatic pressure on monomer P-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy un der pressure and by circular dichroism (CD) and H-1 NMR spectroscopies afte r release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indi cated that at pH 2.0 the recovery of center of spectral mass or ANS fluores cence was almost complete upon pressure release. No difference in H-1 NMR s pectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the c onformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly 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, al though its retinol binding ability was the same as its unpressurized one. F urthermore, CD spectra of the far-UV region and 2D NMR spectra evidently re vealed the difference in the conformation of the molecule between unpressur ized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.