Synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine and its use in the assay of phospholipase A2.

The synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine, 1-O-[12-(2-naphthyl)-dodec-11-enyl]-2-O-decanoyl-sn-glycerol-3- phosphocholine (NVPC), is described. This involves a Wittig reaction between 2-naphthaldehyde and a phosphonium salt which gives the trans-naphthylvinyl group as the predominant isomer. Lyso NVPC was prepared from NVPC by phospholipase A2 action. NVPC absorbs strongly at 248 nm (epsilon = 58,300 M-1 cm-1) and gives broad fluorescence emission with maxima at 343 nm and 360 nm and a quantum yield of 0.10 in ethanol. An assay for phospholipase A2 was developed using high performance liquid chromatography with fluorescence detection to separate and quantify NVPC and lyso NVPC. Activities as low as 1-2 pmol/min in an assay volume of 0.1 ml can easily be measured. The assay was used with a pure enzyme from cobra venom and a crude enzyme from synovial fluid. Enzyme specificities for phosphatidylcholine and NVPC with cobra venom and porcine pancreatic phospholipases A2 were compared using a titrametric assay. The use of the assay with NVPC to study the metabolism of platelet activating factor is discussed.