STRUCTURE-ACTIVITY RELATIONSHIP STUDIES ON ESCHERICHIA COLI BETA-GALACTOSIDASE IN HETEROGENEOUS MEDIA

JULIETA M. SANCHEZ; MARÍA A. PERILLO.

Rio de Janeiro. Brasil. Argentina

Workshop; 2º International Workshop on Spectroscopy for Biology; 2004

NRC,Canada; UFRJ, USP

In the present work we investigated possible correlations between the modulation of b-galactosidase (b-Gal) activity, in the presence of molecular self-assemblies of different chemical composition, supramolecular organization, topology and surface charge, with changes in the protein conformation, by means of intrinsic fluorescence (IF) measurements.             Emission spectra of b-Gal were recorded in the range 300-450 nm, (lex= 295 nm), in the presence or absence of phosphatidylcholine multilamellar vesicles (MLVs) and sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium (CTAB) micelles. Inner filter effects of samples containing MLVs were corrected mathematically. Dynamic quenching of IF was evaluated using acrylamide. Temperature (27-60¢XC) effects on b-Gal structure were also evaluated.             In the presence of SDS and CTAB the intensity of b-Gal IF emission decreased and the lmax was red shifted. An abrupt decrement in IF occured at the cmc of CTAB. MLVs-enzyme interaction induced a decrease in the lmax and an increase in b-Gal IF values. From the analysis of acrylamide-induced quenching of the b-Gal IF emission, Stern-Volmer constants values were determined: Ksv/water= 6.5 and Ksv/MLVs= 0.15 in the presence and absence of MLVs, respectively. The thermal analysis of b-Gal IF in the presence of MLVs showed that the decrease in the IF is lower respect to that measured without lipids (34% and 50% respectively). Thermal denaturation of b-Gal was irreversible in the presence of SDS but CTAB-enzyme interaction showed a true reversible process.             In the presence of MLVs tryptophan residues (Trp) might be localized more deeply of in the protein core or occluded in the dehydrated membrane-protein interface. In the presence of detergents b-Gal suffered an unfolding that explained the activity impairments described previously. The interaction with MLVs seemed to confer higher structural stability to b-Gal, preventing its temperature-induced denaturation and increasing its activity in normal conditions. We conclude that b-Gal structure is affected in a direction dependent on the supramolecular organization of the interacting interfaces.Supported by CONICET, SeCyT-UNC and ACC.