Rapid enzyme-induced hydrolysis of microgram amounts of phosphatidylcholine on phospholipase A2/celite columns

A method has been developed to hydrolyze microgram amounts of phosphatidylcholine (PC) regiospecifically. Hydrolysis of thesn-glycerol 2-acyl group occurs rapidly on microcolumns of immobilized phospholipase A2 on Celite 545 diatomaceous earth. Close to 90% reaction occurs within the first 5 min. Acyl group analysis then may be accomplished by gas liquid chromatography (GLC) of the resulting fatty acids. Hydrolysis of α-16∶0-β-14∶0 PC demonstrated consistent selectivity for 14∶0 liberation, with small amounts of 16∶0 probably indicative of acyl scrambling during the synthesis of the PC. Hydrolysis of an equal weight mixture of (14∶0)2 PC and (18∶2)2PC demonstrated nonpartiality of the immobilized enzyme for either a saturated or unsaturated substrate. The new methodology offers a convenient and sensitive alternative to the presently used procedures.