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Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A 2 (PLA 2 s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA 2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA 2 s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA 2 s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA 2 -like. Acidic PLA 2 s catalyzed the degradation of all substrates evaluated; however, for the basic PLA 2 s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA 2 s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum , while the basic PLA 2 s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA 2 and Lys49-PLA 2 were associated with one another, denoting synergistic action between these PLA 2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA 2 s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents. [Display omitted] • Nine PLA 2 s isoforms were isolated from B. asper venom. • Four acidic and five basic isoforms of PLA 2 s were characterized. • Five of the isolated PLA 2 s represent unpublished isoforms. • The basic isoforms of PLA 2 s have an antiparasitic effect against P. falciparum. • Synergism between basic isoforms of PLA 2 s increases antiplasmodial effect. [ABSTRACT FROM AUTHOR]