Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis

A new PLA₂ (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE ∼14 KDa and confirmed by MALDI-TOF (14321.98 Da). The enzyme showed K_m 6.0 mM, V_max 3.44 nmol/min, optimum pH was 8.0 and temperature was between 30 and 45 °C, and it had a strict requirement of Ca²⁺ for its activity. The N-terminal sequence of PLA₂ was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA₂ from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA₂ from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD₅₀ than crotoxin complex from C. d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5 μg/ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes.