INFLUENCE OF POLYOLS ON THE STRUCTURAL-PROPERTIES OF KLUYVEROMYCES-LACTIS BETA-GALACTOSIDASE UNDER HIGH HYDROSTATIC-PRESSURE

The conformational changes in dimeric Kluyveromyces lactis P-galactosi dase induced by hydrostatic pressure were investigated by means of its intrinsic tryptophan fluorescence. At high pressure, the fluorescence emission spectrum was shifted to the red, indicating the exposure of buried Trp residues to the aqueous solvent. This spectral change was p aralleled by a loss of enzyme activity. The shift of the emission spec trum was quantified by evaluating the centre of spectral mass ([v(g)]) , which is an intensity-weighted mean wavenumber. The experimental dat a could be fitted to a two-state transition (native-->denatured), corr ected for a linear pressure dependence of [v(g)], and allowed the dete rmination of thermodynamic parameters Delta G(app)(0), V-app and P-1/2 . The results were consistent with a partial unfolding of the protein and not simply with dissociation of this dimeric enzyme. In the presen ce of polyols, the native conformation of beta-galactosidase was consi derably more resistant to pressure. This protective effect of polyols is probably due to a reduced accessibility of water inside the protein structure, through the direct or indirect action of these additives o n the enzyme.