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 -(N-4-nitrobenzo-2-oxo-1,5-di azole)aminododecanoyl Phosphatidylethanolamine is accompanied by an in crease in fluorescence intensity that is linearly proportional to enzy me activity. Substrate is prepared in the absence of detergents as a s onicated dispersion in aqueous buffer. Hydrolysis of the corresponding phosphatidylcholine derivative is more than an order of magnitude slo wer under identical assay conditions. A plot of initial rate versus su bstrate concentration could be fit to a simple Michaelis-Menten relati onship with K-m = 13 mu M. In contrast to commonly used radiochemical assays for this enzyme, the method described here is continuous and al lows estimation of enzyme activity without separation of substrate fro m product. Thus, the method is suitable for both kinetic analysis and large-scale screening using automated readers for 96-well tissue cultu re plates. The fluorescence-based assay displays advantages over other continuous assays for human Type II Phospholipase A(2) based on (a) h igh sensitivity and (b) the use of a commercially available substrate. (C) 1994 Academic Press, Inc.