Development of high-throughput screening assays for profiling snake venom phospholipase A2 activity after chromatographic fractionation. (September 2020) - Still, Kristina B.M.
Abstract: Many organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2 s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2 s 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 PLA2 s 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 PLA2 s. 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 . Highlights: A nanofractionation analytics platform for Phospholipase A2 profiling was developed. Chromatography, nanofractions, bioassays, and mass spectrometry were used. Crude snake venom of 6 species were screened for Phospholipase A2 activity. Snake venoms showed multiple Phospholipase A2-like activities.