CollinLAAO: uma nova L-aminoácido oxidase com potencial leishmanicida isolada da peçonha de Crotalus durissus collilineatus

Abstract

SV-LAAOs (Snake Venom L-amino acid oxidases) are flavoenzymes that catalyse the stereospecific oxidative deamination of L-amino acids into α-keto acids releasing ammonia and hydrogen peroxide as products. These enzymes are the target of numerous researches due to their biological and pharmacological effects that include bactericidal, antiviral, antitumor and antiparasitic effects, attributed mainly to the formation of hydrogen peroxide. The objective of this work was to isolate a new L-amino acid oxidase from the Crotalus durissus collilineatus venom, to characterize it biochemically, as well as to evaluate its leishmanicidal potential on different species of Leishmania spp. The new LAAO, called CollinLAAO, was isolated by two chromatographic steps on gel filtration followed by anionic exchange. This enzyme represented 2.5% of the whole venom, with a molecular mass of 63.1 kDa by SDS-PAGE. CollinLAAOs was stored in AMIB buffer 10 mM at 4ºC protected from light and showed an activity of 3000 U / mg / min, with a 50% reduction after 21 days. CollinLAAO demonstrated higher affinity for the amino acid L-Leucine, and better enzymatic activity at neutral and basic pH at temperatures ranging from 0 to 25 ºC. Its activity was completely inhibited by catalase, as well as in the presence of Na+ and K+. The cytotoxicity tests revealed IC50 of 62.73, 45.05 and 11.66 μg/mL for Leishmania (V.) braziliensis, Leishmania (L.) amazonensis and Leishmania (L.) infantum respectively, the enzyme cytotoxicity was completely inhibited by catalase. CollinLAAO (0.78 μg/mL) significantly increased the intracellular concentration of reactive oxygen species (ROS) and reduced the mitochondrial potential of Leishmania (L.) amazonensis (1.56 and 0.78 μg/mL) and Leishmania (L.) amazonensis (1.56, 0.78 and 0.39 μg/mL). CollinLAAO showed higher toxicity for RAW 264.7 macrophages with an IC50 of 0.55μg/mL, as well as decreased the infectivity index to 26.41% and 31.85% at 0.78 μg/mL and 0.39 μg/mL, respectively, indicating that subtoxic concentrations of CollinLAAO are capable of interfering in vital processes of leishmanias. Thus, the results obtained for CollinLAAO provide important subsidies for the development of therapeutic strategies with leishmanicidal action.