A Continuous Fluorescence Displacement Assay for Phospholipase A2 Using Albumin and Medium Chain Phospholipid Substrates

Abstract

A continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino) undecanoic acid from albumin by decanoic acid released as a result of phospholipase A2-catalyzed hydrolysis of didecanoyl-phosphatidyicholine. Using the phospholipase A2 from Naja naja, this assay will detect activity from 1 ng of pure enzyme and a linear response in terms of fluorescence change with time is observed up to about 50 ng of enzyme. The assay is compared with the original fluorescence displacement assay (Wilton, D.C., 1990, Biochem. J. 266, 435-439) which uses rat liver fatty acid binding protein instead of albumin and is able to utilize any natural long chain phospholipid as substrate. The present assay will provide a very convenient method for detection during enzyme purification of the low molecular weight secreted phospholipase A2 and other phospholipases A that can hydrolyze medium chain phospholipids. The assay should also allow the identification of inhibitors of these enzymes. Other substrates including dioleoylphosphatidylcholine and 1-stearoyl-2-arachidonoyl-phosphatidylcholine were also evaluated in the assay.