Spectroscopic properties of horse liver alcohol dehydrogenase in reversed micellar solutions.
Catalytic and spectroscopic properties of alcohol dehydrogenase from horse liver, incorporated in reversed micellar media, have been studied. Two different reversed micellar systems have been used, one containing an anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT], the other containing a cationic (cetyltrimethylammonium bromide, CTAB) surfactant. With 1-hexanol as substrate the turnover number of the enzyme in AOT-reversed micelles is strongly dependent on the water content of the system. At low wo ([H2O]/[surfactant]) (wo less than 20) no enzymatic activity can be detected whereas at high wo (wo = 40) the turnover is only slightly lower than in aqueous solution. In CTAB-reversed micelles the dependence of the turnover number on wo is much less. The enzymatic activity is in this case significantly lower than in aqueous solution and increases only slightly with an increasing water content of the reversed micelles. Possible interactions of the protein with the surfactant interfaces in the reversed micellar media were studied via circular dichroism and fluorescence measurements. From the circular dichroism of the protein backbone it is observed that the protein secondary structure is not significantly affected upon incorporation in the reversed micelles since the far-ultraviolet spectrum is not altered. Results from time-resolved fluorescence anisotropy experiments indicate that, especially in AOT-reversed micelles, interactions between the protein and the surfactant interface are largely electrostatic in nature, as evident from the dependence on the pH of the buffer used. In CTAB-reversed micellar solutions such interactions appear to be much less pronounced than in AOT.
Vos
K
K
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Laane
C
C
Van Hoek
A
A
Veeger
C
C
Visser
A J
AJ
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D005454:Fluorescence Polarization; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008823:Micelles; D011487:Protein Conformation
D008823:Micelles; D000426:Alcohol Dehydrogenase