CONICET | Buscador de Institutos y Recursos Humanos

In previous works we demonstrated that b-Gal/lipid interaction could modulate the enzyme activity. This modulation could be exerted by a change in the intrinsic reaction mechanism of b-Gal catalyzed reaction as well as in the protein conformation. We showed that enzyme-lipid interaction protected the enzyme from its irreversible inactivation at high temperatures (above 40¢XC). In the present work we studied the correlation between the preservation of the enzyme activity and the conformational stability in the presence of phosphatidyl choline multillamelar vesicles (MLVs). Protein conformation studies in the presence or absence of MLVs were performed using intrinsic fluorescence (IF) measurements and differential scanning calorimetry. Emission spectra of the enzyme in the presence of MLVs showed a decrease in lmax and an increase in the fluorescence intensity, respect to what was observed with free enzyme. Inner filter effects of these samples were corrected mathematically. From the data of acrylamide-induced quenching of b-Gal IF emission, Stern-Volmer constants values were determined: KSV/water=6.5 and KSV/MLVs=0.15 with or without MLVs, respectively. Thermal effects analysis of b-Gal IF showed that the decrease of IF was lower in the presence of MLVs respect to that measured without lipids (34% and 50%, respectively). DSC thermograms showed a non two-state thermal denaturation of the enzyme both free and bound to MLVs. However, the thermal transition seemed to be less cooperative and the aggregating temperature of the unfolded b-Gal increased in the presence of MLVs. These results suggested that, when the protein was bound to MLVs, the tryptophan residues were localized more deeply in the protein core or occluded in the dehydrated lipid-protein interface. This conformational state and/or localization of the enzyme would prevent its thermal inactivation. Supported by CONICET, SeCyT-UNC and ACC

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