Abstract
A simple and highly sensitive method for the measurement of lipase activity has been developed using a fluorescent labeling reagent, 9-bromomethylacridine (9-BMA). With an organic solvent-soluble lipase prepared with synthetic detergent, triglycerides were hydrolyzed in a homogeneous solvent system (such as aqueous tetrahydrofuran). The enzyme reaction was terminated by the addition of DMSO containing 9-BMA; then the liberated fatty acid was labeled in the following derivatization reaction at room temperature for 40min. The 9-acridinylmethyl derivative of the fatty acid was separated and determined by high performance liquid chromatography (HPLC). The sensitivity of the method was so high that several nmol level of fatty acid in 200μl of the reaction mixture could be determined. The effects of the pH and of the concentration of buffer on the fluorescent labeling reaction were examined. This method was found to be useful for studying the kinetic properties of lipase.
