Development of high-throughput screening assays for profiling snake venom phospholipase A₂ activity after chromatographic fractionation

Many organisms, ranging from plants to mammals, contain phospholipase A₂ enzymes (PLA₂s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA₂s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA₂s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA₂ activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA₂s. The methodology was demonstrated for assessment of PLA₂ activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.