A continuous fluorescence assay for phospholipase A2 with nontagged lipid.

Human nonpancreatic secreted phospholipase A2 (hnps PLA2) is considered to be an important drug target for antiinflammation therapy. We have established a new fluorescence assay by using 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial probe for hydrophobic environment detection. The fitted apparent k(cat)/K(m) of hnps PLA2 is 0.0181 +/- 0.0005 RFU/microMs. Tests on known synthesized inhibitor gave IC50 values similar to those from isotope-labeled assay. Because ANS is a commonly used probe for hydrophobic environment detection that needs no modification in the current assay, this strategy may be widely applicable for interfacial catalytic reactions.