A FLUORESCENCE-BASED ASSAY FOR HUMAN TYPE-II PHOSPHOLIPASE A(2)

A fluorescence assay for quantitation of human Type II Phospholipase A(2) activity is described. Hydrolysis of 1-Acyl-2-(N-4-nitrobenzo-2-oxo-1,5-diazole)aminododecanoyl Phosphatidylethanolamine is accompanied by an increase in fluorescence intensity that is linearly proportional to enzyme activity. Substrate is prepared in the absence of detergents as a sonicated dispersion in aqueous buffer. Hydrolysis of the corresponding phosphatidylcholine derivative is more than an order of magnitude slower under identical assay conditions. A plot of initial rate versus substrate concentration could be fit to a simple Michaelis-Menten relationship with K-m = 13 mu M. In contrast to commonly used radiochemical assays for this enzyme, the method described here is continuous and allows estimation of enzyme activity without separation of substrate from product. Thus, the method is suitable for both kinetic analysis and large-scale screening using automated readers for 96-well tissue culture plates. The fluorescence-based assay displays advantages over other continuous assays for human Type II Phospholipase A(2) based on (a) high sensitivity and (b) the use of a commercially available substrate. (C) 1994 Academic Press, Inc.