A subnanogram assay for phospholipase activity based on a long-chain radioiodinatable phosphatidylcholine.

CARAMELO JJ; DELFINO JM

ANALYTICAL BIOCHEMISTRY

Año: 2004 vol. 333 p. 289 - 295

0003-2697

Here, we introduce a radioiodinatable long-chain phosphatidylcholine (BHC12PC) which serves as the base for a very sensitive phospholipase assay. This compound has a 4-hydroxyphenyl group attached at the end of the fatty acyl chain located in position sn-2. This feature enables this phospholipid to be radioiodinated. BHC12PC was tested as a substrate of Naja naja naja PLA(2) and Bacillus cereus PLC in a mixed micellar system with Triton X-100. The detection limit for the assays was 0.25ng of PLA(2) and 0.05ng of PLC, thus becoming one of the most sensitive methods described so far. A low specific radioactivity (500microCi/mmol) suffices to achieve this level of sensitivity. In both cases, the behavior of BHC12PC was indistinguishable from that shown by phospholipids with n-acyl chains of similar length. The choice of spacer prevents any unfavorable interaction of the bulky 4-hydroxyphenyl group at the active site of the enzymes. The progress of the reaction as monitored by thin-layer chromatography is compared side by side with an alternative method based on the selective adsorption of BHC12PC to silica gel, which renders identical results in a simpler fashion. An additional advantage of BHC12PC is that the cost per Ci of the radioiodinated derivative is significantly lower than that of other labeled phospholipids ((3)H, (14)C, or (32)P).