Or A2 Activities using Synthetic Phosphatidylcholines containing -eleostearic Acid Coated on Microtiter Plates

Development of a Continuous and High throughput Assay for Measuring Phospholipase A₁ or A₂ Activities using Synthetic Phosphatidylcholines containing -eleostearic Acid Coated on Microtiter Plates

Meddy El Alaoui^1,2,,Laurent Soulere²_, Alexandre Noiriel¹, Florence Popowycz², Yves Queneau² and Abdelkarim Abousalham<sup>1

1</sup>Organisation et Dynamique des Membranes Biologiques, ICBMS UMR 5246 CNRS Université Lyon 1, Bât Raulin, 43 Bd du 11 Novembre 1918. 69621 Villeurbanne Cedex, France

²Chimie Organique et Bioorganique, ICBMS UMR 5246 CNRS INSA Lyon, Bât Jules Verne, 20 av. A. Einstein, 69621 Villeurbanne Cedex, France
To date, several sensitive methods based on radiolabeled elements or sterically hindered fluorochrome groups are usually employed to screen phospholipase A₁ (PLA₁) and A₂ (PLA₂) activities. Here, to develop a new ultraviolet (UV) spectrophotometric assay for PLA₁ and PLA₂, we have synthesized specific phosphatidylcholines (PC) esterified at the sn-1 and/or sn-2 positions with -eleostearic acid (9Z, 11E, 13E octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in -eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on this free fatty acid as well as of the PC harboring it. When coated on the surface of a microtiter 96-well plate these compounds enable the screening of PLA₁ or PLA₂ activities. Upon enzyme injection at the oil/water interface, -eleostearic acid is release, solubilized with -cyclodextrin and the UV absorbance is considerably enhanced due to its transition from the adsorbed state to the soluble state. This phospholipase assay is based on the difference between the apparent molar extinction coefficients of the two types of -eleostearic acid either esterified into PC or free into the reaction medium. Consequently, the phospholipase activity can be followed continuously by spectrophotometry using the UV absorption spectrum. The rate of lipolysis was monitored by measuring the increase of optical density at 272 nm, which was found to be linear with time and amount of added enzyme. This method could be used to differentiate PLA₁ and PLA₂ activities. Moreover, this method allows screening new PLA₁ and PLA₂ activities or their inhibitors present in various biological samples.