A continuous spectrophotometric assay that distinguishes between phospholipase A(1) and A(2) activities

A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A(1) (PLA(1)) or phospholipase A(2) (PLA(2)) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA(1) or PLA(2) activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA(1) and PLA(2) activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA(1) enzyme), bee venom PLA(2), and porcine pancreatic PLA(2) were all used to validate the assay. Compared with current assays used for continuously measuring PLA(1) or PLA(2) activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.