Isolation and characterization of two phospholipases A2 isoforms from Crotalus durissus terrificus venom from Argentina

TORRES-HUACO, FANK; RODRIGUEZ JUAN PABLO; FUSCO LUCIANO; BUSTILLO SOLEDAD; MARANGONI SERGIO; ACOSTA, OFELIA; LEIVA LAURA CRISTINA; PONCE-SOTO LUIS

Paraná

Congreso; XLI Annual Meeting of The Brazilian Biochemistry and Molecular Biology Society; 2012

Two basic proteins with phospholipase A2 activity, designated P9(Cdt-PLA2) and P10(Cdt-PLA2) were purified from the venom of the Northeast Argentinian viperid Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly showed that P9(Cdt-PLA2) is 2.5 times more abundant than P10(Cdt-PLA2). The molecular mass was determined by mass spectrometry resulting in 14181.31 for P9(Cdt-PLA2) Da and 14231.78 Da for P10(Cdt-PLA2), both of them displayed a monomeric conformation. The kinetic assay developed showed a catalytically active phospholipase A2 in correspondence with Asp49 PLA2 group, as expected with Km: 1.34.10-3 M and Vmax: 28.69 nmol/min for P9(Cdt-PLA2) and Km: 1.15.10-3 M and Vmax: 33.5 nmol/min for P10(Cdt-PLA2). P9(Cdt-PLA2) and P10(Cdt-PLA2) isoforms contain an amino acid composition of 121 and 122 residues, respectively and a calculated theoretical pI value of 8.47 and 8.67, respectively. Amino acid sequence alignments with other crotalic PLA2 revealed a high degree of indentity in the primary sequence (over 80%). Additionally, we studied the neurotoxicity in muscle preparations of biventer cervis from chicken; edematogenic in footpad and myotoxic activities in gastrocnemius muscle, both in mice, as well as studies of cytotoxicity in cell culture (C2C12, murine myoblast). The results of these trials demonstrated that P9 (Cdt-PLA2) is significantly less toxic than P10(Cdt-PLA2) and both of them show different pharmacological properties compared to those described by other Brazilian authors. We conclude that the most abundant isoform isolated, P9(Cdt-PLA2), was found to have less deleterious effects. The results obtained suggest that this PLA2 has the desired conditions (good purification performance and low toxicity) to be used as an immunogen in the process of obtaining crotalic antitoxins.