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

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...

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Autores principales: Gómez, Gabriela Noemí, Leiva, Laura Cristina Ana, Nerli, Bibiana Beatriz
Formato: Artículo
Lenguaje:Inglés
Publicado: Academic Press Inc Elsevier Science 2021
Materias:
Aqueous
Snake venom
Phospholipase A2
Protease
Acceso en línea:http://repositorio.unne.edu.ar/handle/123456789/30338
Aporte de:
RIUNNE - Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE) de Universidad Nacional del Nordeste
id I48-R184-123456789-30338
record_format dspace
spelling I48-R184-123456789-303382025-03-06T11:04:32Z Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom Gómez, Gabriela Noemí Leiva, Laura Cristina Ana Nerli, Bibiana Beatriz Aqueous Snake venom Phospholipase A2 Protease 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. 2021-12-09T17:02:47Z 2021-12-09T17:02:47Z 2016 Artículo Gómez, Gabriela Noemi,Leiva, Laura Cristina Ana y Nerli, Bibiana Beatriz, 2016. Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom. Protein expression and purification. San Diego: Academic Press Inc Elsevier Science, vol. 124, p. 68-74. ISSN 1046-5928. 1046-5928 http://repositorio.unne.edu.ar/handle/123456789/30338 eng openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ application/pdf application/pdf Academic Press Inc Elsevier Science Protein Expression and Purification, 2016, vol. 124, p. 68-74.
institution Universidad Nacional del Nordeste
institution_str I-48
repository_str R-184
collection RIUNNE - Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
language Inglés
topic Aqueous
Snake venom
Phospholipase A2
Protease
spellingShingle Aqueous
Snake venom
Phospholipase A2
Protease
Gómez, Gabriela Noemí
Leiva, Laura Cristina Ana
Nerli, Bibiana Beatriz
Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom
topic_facet Aqueous
Snake venom
Phospholipase A2
Protease
description 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.
format Artículo
author Gómez, Gabriela Noemí
Leiva, Laura Cristina Ana
Nerli, Bibiana Beatriz
author_facet Gómez, Gabriela Noemí
Leiva, Laura Cristina Ana
Nerli, Bibiana Beatriz
author_sort Gómez, Gabriela Noemí
title Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom
title_short Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom
title_full Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom
title_fullStr Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom
title_full_unstemmed Aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom
title_sort aqueous two-phase systems: a simple methodology to obtain mixtures enriched in main toxins of bothrops alternatus venom
publisher Academic Press Inc Elsevier Science
publishDate 2021
url http://repositorio.unne.edu.ar/handle/123456789/30338
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