Direct observation of the action of cholesterol oxidase in monolayers

Abstract

The oxidation of monolayer cholesterol by cholesterol oxidase has been visualized using monolayer fluorescence microscopy. A direct microscopic visualization was possible because the lateral distribution of a lipid fluorophore, tetramethylrhodamine (TRITC)-labeled phosphatidylethanolamine, was very different in a cholesterol containing monolayer as compared with a cholestenone monolayer. The lipid fluorophore was effectively excluded from the condensed cholesterol phase, but was readily miscible in the cholestenone phase. One could therefore observe the appearance of fluorophore rich cholestenone-domains in the cholesterol monolayer as a result of the cholesterol oxidase catalyzed oxidation reaction. The oxidation experiments were performed at 22°C with a monolayer surface pressure of 5 mN/m (on 50 mM Tris-HCI buffer, containing 140 mM NaCl, pH 7.4). When 40 mU/ml of cholesterol oxidase was injected beneath the monolayer under observation, it appeared that the enzyme penetrated the cholesterol monolayer at random sites and initiated the oxidation reaction. Once the oxidation reaction had commenced, it progressed rapidly and converted the condensed (cholesterol-rich) phase into an expanded (cholestenone-rich) phase. When the oxidation of cholesterol in mixed cholesterol /dimyristoylphosphatidylcholine monolayers was visualized, it was observed that the enzyme-catalyzed oxidation started from the expanded phases (domains with higher compressibility) and the reaction eventually led to the dissipation of the boundary line between expanded and condensed phases. With time all condensed phases were dissolved and the monolayer became uniformly fluorescent. The association of TRITC-labeled cholesterol oxidase with a non-fluorescent mixed cholesterol /dimyristoylphosphatidylcholine monolayer led to the penetration (or association) of the fluorescent cholesterol oxidase into expanded phases of the mixed monolayers. The monolayer lateral domain morphology was similar whether the fluorescent probe was TRITC-PE or TRITC-labeled enzyme. It is concluded that cholesterol oxidase associated with (or penetrated to some extent into) the expanded phases of a monolayer, and carried out its oxidation reaction in the expanded phase or at the interface between expanded and condensed phases.