Abstract

This study reports the purification of ML-LAAO, a new LAAO from the venom of Micrurus lemniscatus snake (ML-V), using size exclusion chromatography. ML-LAAO is a 69-kDa glycoprotein that represents similar to 2.0% of total venom proteins. This enzyme exhibited optimal activity at pH 8.5, displaying high specificity toward hydrophobic L-amino acids. MALDI TOF/TOF and Blast analysis identified internal segments in ML-LAAO that share high sequence identity with homologous snake venom LAAOs. Western blot analysis on two-dimensional SDS-PAGE of ML-V, using anti-LAAO revealed the presence of ML-LAAO isoforms (pI 6.3-8.9). ML-LAAO blocked aggregation induced by collagen on washed platelets in a rather weak manner, it did not, however, inhibit platelet aggregation induced by ADP on platelet-rich plasma. In addition, this enzyme displayed in vitro antibacterial activity against Staphylococcus aureus (MIC/MBC of 0.39 mu g/mL) and in vitro leishmanicidal action against Leishmania amazonensis and L. chagasi (IC50 values of 0.14 and 0.039 mu g/mL, respectively). These activities were significantly reduced by catalase, suggesting that hydrogen peroxide production is involved in some way. The data presented here revealed that ML-LAAO has bactericidal and leishmanicidal effects, suggesting that it may have therapeutic potential. (C) 2019 Elsevier B.V. All rights reserved.