An aqueous endpoint assay of snake venom phospholipase A2

Phospholipase A2 (PLA2), an enzyme found in most snake venoms, catalyzes the hydrolysis of phospholipids in biological membranes, and some have presynaptic neurotoxic activity. A synthetic substrate, 4-nitro-3-(octanoyloxy)benzoic acid, was synthesized and purified on a silica gel column using a published method. This substrate was used to develop an endpoint assay which is rapid and requires a minimum of equipment. This aqueous assay system allowed enzyme activity to be examined without the use of radioactive substrates or organic solvents, minimizing waste disposal concerns. Whole venoms, partially purified enzyme isolated from Crotalus mitchelli pyrrhus venom, tissue extracts and commercial preparations were employed as sources of PLA2. Results show that this method is a convenient and specific assay for PLA2 from several sources and is particularly suited for assaying large numbers of fractions generated during purification procedures.