Influence of polyols on the structural properties of Kluyveromyces lactis beta-galactosidase under high hydrostatic pressure.
The conformational changes in dimeric Kluyveromyces lactis beta-galactosidase 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 paralleled by a loss of enzyme activity. The shift of the emission spectrum was quantified by evaluating the centre of spectral mass ((nu(g))), which is an intensity-weighted mean wavenumber. The experimental data could be fitted to a two-state transition (native<-->denatured), corrected for a linear pressure dependence of (nu(g)), and allowed the determination of thermodynamic parameters deltaG0(app), 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 presence of polyols, the native conformation of beta-galactosidase was considerably 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 on the enzyme.
Institut National des Sciences Appliquées de Toulouse, Centre de Bioingénierie Gilbert Durand, Complexe Scientifique de Rangueil, France.
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D000818:Animals; D005511:Food Handling; D006023:Glycoproteins / Q000737:chemistry* / Q000187:drug effects; D006874:Hydrostatic Pressure; D007716:Kluyveromyces / Q000201:enzymology*; D008892:Milk; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013402:Sugar Alcohols / Q000494:pharmacology*; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000737:chemistry* / Q000187:drug effects
D006023:Glycoproteins; D013402:Sugar Alcohols; D001616:beta-Galactosidase