An improved spectrophotometric phospholipase A2 assay using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate and lipoxygenase as coupled enzyme

Abstract

An improved spectrophotometric assay of phospholipase A2 (PLA2) activity based on the coupled PLA2/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PCLIN) as substrate is reported. The PLA2-mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 (3 μL/μmol phospholipid) as surfactant and of ethanol (15 μL/mL reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory PLA2 from honey bee venom (HBV-PLA2). The enzyme did not discriminate among PCLIN, phosphatidylcholine, and phosphatidylethanolamine, but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine (PCDILIN). Nevertheless, the use of PCDILIN is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine, are known to be oxidized by LOX. HBV-PLA2 showed maximal activity at pH 9.0, hyperbolic kinetics (Km, 74.2±2.9 μM; Vmax, 827±7 μmol/min/mg protein) and competitive inhibition (Ki about 5 μM) by palmityl trifluoromethyl ketone, a classical PLA2 inhibitor. Interestingly, the HBV-PLA2/soybean LOX-1 coupled reactions also allow an accurate assay of PCLIN concentration. In the whole, these results demonstrate that this improved PLA2/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.