ABSTRACT
Components with phospholipase A2 activity were isolated by gel filtration and cationic exchange chromatography from the venom of Bothrops asper snakes from Chiriguaná, Colombia (9°22´N; 73°37´W). Five fractions were obtained by the gel filtration, and PLA2 activity was found in fraction 3 (F3). In the cationic exchange chromatography, F3 showed eight components with PLA2 activity. Six of these components appeared as one band in polyacrylamide gel electrophoresis (SDS-PAGE). Fractions II and VII exhibited an optimal activity at pH 9 and 52ºC. The optimum calcium concentration for fraction II was 48 mM and for fraction VII, 384 mM. Both fractions showed thermal stability. Fraction II was stable at pH values between 2.5 and 9, and fraction VII, between 2.5 and 8. The Michaelis Menten constant (K M) was 3.5x10-3 M for fraction II and 1.6x10-3 M for fraction VII. The molecular weight was 16,000 Dalton for fraction II and 17,000 Dalton for fraction VII. Both isoenzymes did not show any toxic activity (DL50) at 5.3 and 4 µg/g. The two fractions showed different kinetic constant (K M), calcium requirement, and substrate specificity for haemolytic activity.
Subject(s)
Animals , Male , Mice , Phospholipases A , Crotalid Venoms/classification , Crotalid Venoms/isolation & purification , ColombiaABSTRACT
La fosfolipasa A2 (PLA2) fue purificada a partir de vene-no de serpiente de Bothrops atrox (Sensu lato) de Chiriguaná (Colombia) por cromatografía de exclusión sobre Sephadex G-75, de donde se obtuvieron cinco frac-ciones, una de ellas con actividad fosfolipasa A2. Des-pués de pasarla por una columna de intercambio catiónico Mono S, donde se separaron ocho fracciones con activi-dad PLA2, ésta se midió usando el método hemolítico. La electroforesis en gel de poliacrilamida en condiciones desnaturalizantes, mostró pesos moleculares entre 16.000 y 17.000 para las ocho fracciones, seis de las cuales pre-sentaron una sola banda.