Biochemical Diversity of Phospholipases A₂ and Metalloproteinases Isolated from Bothrops leucurus Venom

Snake envenomation by Bothrops species represents a major public health problem in Brazil. Bothrops leucurus, endemic to northeastern Brazil, is not included in the production pool of commercial antibothropic antivenoms, highlighting the need to investigate its biological effects. This study aimed to isolate and biochemically characterize toxins from B. leucurus venom (VBleu). VBleu was subjected to hydrophobic interaction chromatography (Phenyl/HIC), followed by ion-exchange chromatography (Mono Q) and gel filtration. Subsequently, crude venom was submitted to RP-HPLC to isolate an additional Phospholipases A₂ (PLA₂) isoform of the Lys49 type, which was not resolved in previous chromatographic steps. Fractions were analyzed by SDS-PAGE, MALDI-TOF, LC-MS/MS, and fluorometric enzymatic assays (EnzChek PLA₂ and FRET). Snake venom metalloproteinases (SVMPs) belonging to the PIII (~50–60 kDa) and PI (~23 kDa) classes were identified. PIII SVMPs exhibited high proteolytic activity (~800 RFU/min/µg), which was completely inhibited by EDTA, confirming their zinc-dependent catalytic profile. In contrast, PI SVMPs showed low proteolytic activity despite higher relative abundance (42% coverage), highlighting functional differences between classes. Chromatographic steps also revealed fractions with a molecular mass of ~13.6 kDa, high phospholipase activity in the EnzChek PLA₂ assay (~14,000 RFU/min/µg), and 87% proteomic coverage, supporting the presence of a catalytically active Asp49 PLA₂ isoform.RP-HPLC enabled the isolation of an additional PLA₂ (~13.8 kDa) with no detectable phospholipase activity, identified by LC-MS/MS as a Lys49 homolog. Thus, the purification strategy enabled the isolation of the major toxins from B. leucurus venom, providing a basis for biochemical and functional characterization. These fractions allowed direct comparison between PLA₂s and SVMPs, contributing to the understanding of the functional diversity among these toxin classes and supporting the development of more comprehensive therapeutic approaches.