Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

Abstract Venoms from the bee Apis mellifera , the caterpillar Lonomia achelous , the spiders Lycosa sp. and Phoneutria nigriventer , the scorpions Tityus bahiensis and Tityus serrulatus , and the snakes Bothrops alternatus , Bothrops jararaca , Bothrops jararacussu , Bothrops moojeni , Bothrops neuwiedi , Crotalus durissus terrificus , and Lachesis muta were assayed (800 μg/mL) for activity against Staphylococcus aureus . Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca , venom was fractionated through gel exclusion chromatography. The high molecular weight, anti- S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0–0.5 M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l -leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti- S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H 2 O 2 production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l -leucine. The correlation between in vivo neutralization of lethal toxicity (ED 50 ) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus , B. jararacussu , B. neuwiedii and B. moojeni ). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients ( r ) between ED 50 and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 ( p p Leishmania amazonensis ) activity of LAAO was also determined.