A phospholipase A2 kinetic and binding assay using phospholipid-coated hydrophobic beads

A novel kinetic and membrane-binding assay for phospholipase A2 (PLA2) has been developed utilizing phospholipid-coated hydrophobic styrene-divinylbenzene beads (5.2 +/- 0.3 microm diameter). Phospholipids formed a stable monolayer film on styrene-divinylbenzene beads with average surface packing density of (1.3 +/- 0.2) x 10(-2) molecule/A2. Secretory PLA2 readily hydrolyzed 1-palmitoyl-2-[3H]-oleoyl-sn-glycero-3-phosphoglycerol coated on styrene-divinylbenzene beads which could be easily monitored by measuring the radioactivity of fatty acid released to solution in the presence of bovine serum albumin. For human cytosolic PLA2 with high specificity for sn-2 arachidonyl group, styrene-divinylbenzene beads coated with 1-stearoyl-2-[14C]-arachidonyl-sn-glycero-3-phosphocholine and dioleoylglycerol (7:3, mol/mol) were used as substrate. PLA2 activity was linearly proportional to the enzyme concentration in the range from 1 to 150 nM for human class II secretory PLA2 and from 1 to 20 nM for cytosolic PLA2; the specific activity was 1.6 and 1.7 micromol/min/mg, respectively. Finally, styrene-divinylbenzene beads coated with polymerized 1,2-bis[12-(lipoyloxy) dodecanoyl]-sn-glycero-3-phosphoglycerol were used to measure the membrane binding affinity of PLA2, which in conjunction with kinetic data provides important insights into how PLA2 interacts with membranes.