Use of a phosphopeptide as a ligand to purify phospholipase A2 from the venom of Crotalus durisuss terrificus by affinity chromatography

SAAVEDRA, SOLEDAD L.; ACOSTA, GERARDO; AVILA, LUCÍA; GIUDICESSI, SILVANA L.; CAMPERI, SILVIA A.; ALBERICIO, FERNANDO; CASCONE, OSVALDO; MARTINEZ CERON, MARIA C.

JOURNAL OF CHROMATOGRAPHY B

ELSEVIER

Lugar: Amsterdam; Año: 2020

0378-4347

The venom of the South American snake Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Among them, the PLA2 subunit of crotoxin (Ctx) has been studied for its possible use as an antiviral, antibacterial and antitumoral agent. Peptides have proven to be very useful ligands for the purification of numerous molecules such as antibodies, toxins, enzymes, among other proteins. Here, we aimed to use a phosphopeptide (P-Lys) as a ligand for PLA2purification. The synthesis was carried out on solid phase on Rink-Amide-ChemMatrix resin and then P-Lys was immobilized on NHS-agarose and evaluated as a chromatographic matrix. Under the best conditions, the percentage of total protein adsorption reached 39% and only the eluate fraction presented PLA2 enzymatic activity. The analysis by SDS-PAGE of the eluate showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 oligomers. The yield of the process exceeded 100% and the purity was over 90%. In addition, the process allows isolation of crotamine.