title: Aqueous two-phase systems: A simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom.
abstract: 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.
authors.affiliation: Laboratorio de Investigación en Proteínas (LabInPro), Instituto de Química Básica y Aplicada del Nordeste Argentino (IQUIBA-NEA) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste (UNNE), Av. Libertad 5470, Corrientes 3400, Argentina.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Gomez
authors.affiliation: Laboratorio de Investigación en Proteínas (LabInPro), Instituto de Química Básica y Aplicada del Nordeste Argentino (IQUIBA-NEA) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste (UNNE), Av. Libertad 5470, Corrientes 3400, Argentina.
authors.forename: Laura
authors.initials: L
authors.lastname: Leiva
authors.affiliation: Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, S2002LRK Rosario, Argentina. Electronic address: bnerli@fbioyf.unr.edu.ar.
authors.forename: Bibiana Beatriz
authors.initials: BB
authors.lastname: Nerli
mesh_terms: D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000737:chemistry*; D003636:DEAE-Cellulose; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D003435:Crotalid Venoms; D011092:Polyethylene Glycols; D003636:DEAE-Cellulose; D054467:Phospholipases A2; D012697:Serine Endopeptidases; C000595209:polyethylene glycol 1000
keywords: Aqueous two-phase systems; Phospholipase A(2); Protease; Snake venom