CONTINUOUS MEASUREMENT OF PHOSPHOLIPASE A(2) ACTIVITY USING THE FLUORESCENT-PROBE ADIFAB

A new method is described for the continuous quantitation of phospholi pase A(2) (PLA(2)) activity with greatly improved sensitivity compared to existing techniques. The method utilizes a fluorescent probe to de tect the release of fatty acid monomers (free fatty acids) into the aq ueous phase. The fluorescent probe ADIFAB, which is the acrylodan deri vative of rat intestinal fatty acid binding protein, exhibits a change in the ratio of its fluorescence upon binding medium- to long-chain n ative fatty acids. ADIFAB was used to measure the hydrolysis of artifi cial and natural membranes using PLA(2)s from porcine pancreas, Naja m ocambique mocambique, Crotalus durissus terrificus, and bee venom. Tot al phospholipid hydrolysis was determined from the free fatty acid con centration using membrane/water partition coefficients, also measured using ADIFAB, The results indicate that continuous monitoring of natur al 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 mo st commonly used pH stat method. (C) 1995 Academic Press, Inc.