Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom

• Autores: Gómez, Gabriela Noemí; Leiva, Laura Cristina Ana; Nerli, Bibiana Beatriz
• Año de publicación: 2016
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Protein Expression and Purification, 2016, vol. 124, p. 68-74.
• Materia: Aqueous; Snake venom; Phospholipase A2; Protease
• Institución: Universidad Nacional del Nordeste

Fil: Gómez, Gabriela Noemí. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura; Argentina.
Fil: Gómez, Gabriela Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina.
Fil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura; Argentina.
Fil: Leiva, Laura Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina.
Fil: Nerli, Bibiana Beatriz. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.
Fil: Nerli, Bibiana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina.
Phospholipase A2 (PLA2) and protease (P) are enzymes responsible of myotoxic, edematogenic and hemostasis disorder effects observed in the envenomation by Bothrops alternatus pitviper. Their partitioning coefficient (Kp) in different polyethyleneglycol/potassium phosphate aqueous two-phase systems (ATPSs) was determined in order to both achieve a better understanding of the partitioning mechanism and define optimal conditions for toxin isolation. Polyethyleneglycols (PEGs) of molecular weights 1000; 3350; 6000 and 8000; different temperatures (5, 20 and 37 C) and phase volume ratios of 0.5; 1 and 2 were assayed. PLA2 partitioned preferentially to the top phase while P mainly distributed to the bottom phase. Either entropically- or enthalpically-driven mechanisms were involved in each case (PLA2 and P). The aqueous two-phase system formed by PEG of MW 3350 (12.20% wt/wt) and KPi pH 7.0 (11.82% wt/ wt) with a volume ratio of one and a load of 1.25 mg of venom/g of system showed to be the most efficient to recover both enzymes. It allowed obtaining the 72% of PLA2 in the top phase with a purification factor of 2 and the 82% of P at the bottom phase simultaneously. A further adsorption batch step with DEAE-cellulose was used to remove satisfactorily the PEG from the top phase and recover the active PLA2. The proposed methodology is simple, inexpensive, and only requires professionals trained in handling basic laboratory equipment. It could be easily adoptable by developing countries in which the snakebite accidents cause considerable morbidity and mortality.