Isolation of a phospholipase A2 from Bothrops alternatus venom

GOMEZ, GABRIELA; NERLI, BIBIANA; ACOSTA, OFELIA; PICÓ, GUILLERMO; LEIVA, LAURA

Rosario, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular – (SAIB); 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular – (SAIB)

Snake venoms toxins are frequently isolated by chromatographic methods. Aqueous two-phase systems have been successfully used for separation and purification of macromolecules because exhibit multiple advantages: good resolution, high yield, low cost and the protein remain their biological activity. In this work we applied a polyethyleneglycol (PEG)-phosphate-water system to isolate a phospholipase A2 (PLA2) from Bothrops alternatus (yarará) venom. A two-phase system forming by PEG 3350 - potassium phosphate, pH 7.0, was used where the partition of crude venom proteins were assayed. After mixing through inversion and leaving it to settle, the system was centrifuged for the two-phase separation. The top phase was removed and replaced by pure top phase; then, the partition was carried out again. Samples of each phase were withdrawn and their protein content and phospholipase, coagulant and proteolitic activities were assayed. Three partitions, that included the corresponding top phase renovations, were required to isolated the PLA2 in the bottom phase. Another protein presents in this phase was removed by ultrafiltration (30 kDa). SDS-PAGE electrophoresis of the isolated enzyme showed a single typical PLA2 band of 14,8 kDa. We concluded that this partition procedure constitutes a viable procedure for isolation and purification of PLA2 from bothropic venom. Key words: phospholipase A2 - aqueous biphasic system - Bothrops alternatus - polyethyleneglycol