8] Phospholipase A2 assays with fluorophore-labeled lipid substrates

Abstract

Fluorophore-labeled lipid substrates are used in two different ways to assay phospholipase A2 (PLA2). Continuous assays utilize changes in the fluorescence properties of the fluorophore on hydrolysis. These two methods involve chromatographic separation of fluorescent substrate and product by high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) and direct determination of the ratio of substrate to product by fluorescence detection. The fluorophore-labeled lipid substrates used in these assays have a fluorophore attached to the end of a hydrocarbon chain linked to the glycerol backbone by an ether linkage at the sn-1 position. Normal fatty acids are esterified at the sn-2 position. These substrates are resistant to phospholipase A1 action, and the products are resistant to lysophospholipase action, making the assay quite specific for PLA2. The phospholipid substrates and lysophospholipid products, both fluorescent, are separated by HPLC or TLC. Naphthylvinyl-PC (NVPC) is used in the HPLC assay, which absorbs strongly at 250 nm and emits above 340 nm. The HPLC and TLC assays are sensitive enough to detect low levels of PLA2 in crude physiological sources such as synovial fluid. The TLC assay is suitable for rapid and efficient screening of PLA2 inhibitors since various assays can simultaneously be analyzed on a single TLC plate. Qualitative results may be obtained by simple viewing of the plate under UV light for dansyl-PC or ordinary light for dabsyl-PC.