A fluorescence-based assay for human type II phospholipase A2.
A fluorescence assay for quantitation of human Type II Phospholipase A2 activity is described. Hydrolysis of 1-Acyl-2-(N-4-nitrobenzo-2-oxo-1,3-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 Km = 13 microM. 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 A2 based on (a) high sensitivity and (b) the use of a commercially available substrate.
Blanchard
S G
SG
Department of Biochemistry, Glaxo Research Institute, Research Triangle Park, North Carolina 27709.
Harris
C O
CO
Parks
D J
DJ
D005470:Fluorometry / Q000379:methods*; D006801:Humans; D016014:Linear Models; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D013379:Substrate Specificity; D017186:Titrimetry
D010741:Phospholipases A; D054467:Phospholipases A2