Continuous measurement of phospholipase A2 activity using the fluorescent probe ADIFAB

A new method is described for the continuous quantitation of phospholipase A2 (PLA2) activity with greatly improved sensitivity compared to existing techniques. The method utilizes a fluorescent probe to detect the release of fatty acid monomers (free fatty acids) into the aqueous phase. The fluorescent probe ADIFAB, which is the acrylodan derivative of rat intestinal fatty acid binding protein, exhibits a change in the ratio of its fluorescence upon binding medium- to long-chain native fatty acids. ADIFAB was used to measure the hydrolysis of artificial and natural membranes using PLA2s from porcine pancreas, Naja mocambique mocambique, Crotalus durissus terrificus, and bee venom. Total phospholipid hydrolysis was determined from the free fatty acid concentration using membrane/water partition coefficients, also measured using ADIFAB. The results indicate that continuous monitoring of natural substrates can be determined with a sensitivity limit of less than 1 pmol/min, a more than 10(4)-fold increase in sensitivity over the most commonly used pH stat method.