Use of a phosphopeptide as a ligand to purify phospholipase A 2 from the venom of Crotalus durisuss terrificus by affinity chromatography

The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A (PLA ) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.