Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes

We report on the determination of active enzyme components in pure and crud e lipases, using fluorescent inhibitors for covalent modification and visua lization of the enzymatically active proteins. Lipase-specific compounds ar e triacylglycerol analogs, namely 1,2(2,3)-di- O-alkylglyceroalkylphosphoni c acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcoh ols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible tow ard nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protei n complexes are formed which can be analyzed on the basis of their fluoresc ent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the a nalysis of a commercial lipase preparation from Rhizomucor miehei. After in cubation of the enzyme with the fluorescent lipid, a single fluorescence ba nd was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtai ned between fluorescence intensity and the amount of enzyme. Using a combin ation of different inhibitors, we were able to discriminate between lipases and esterases. (C) 1999 Academic Press.