Biochemical and biological characterization of Bothriechis schlegelii snake venoms from Colombia and Costa Rica

Abstract

Snakebites inflicted by the arboreal viperid snake Bothriechis schlegelii in humans are characterized by pain, edema, and ecchymosis at the site of the bite, rarely with blisters, local necrosis, or defibrination. Herein, a comparative study of Bothriechis schlegelii snake venoms from Colombia (BsCo) and Costa Rica (BsCR) was carried out in order to compare their main activities and to verify the efficacy of Bothrops antivenom produced in Brazil to neutralize them. Biochemical (SDS-PAGE and zymography) and biological parameters (edematogenic, lethal, hemorrhagic, nociceptive, and phospholipase A activities) induced by BsCo and BsCR snake venoms were evaluated. The presence of antibodies in Bothrops antivenom that recognize BsCo and BsCR snake venoms by enzyme-linked immunosorbent assay and Western blotting, as well as the ability of this antivenom to neutralize the toxic activities were also verified. SDS-PAGE showed differences between venoms. Distinctive caseinolytic and hyaluronidase patterns were detected by zymography. BsCo and BsCR showed similar phospholipase A activity. Strong cross-reactivity between BsCo and BsCR was detected using Bothrops antivenom with many components located between 150 and 35 kDa. BsCR was more edematogenic and almost fourfold more hemorrhagic than BsCo, and both venoms induced nociception. BsCR (LD 5.60 mg/kg) was more lethal to mice than BsCo (LD 9.24 mg/kg). Bothrops antivenom was effective in the neutralization of lethal and hemorrhagic activities of BsCo and BsCR and was partially effective in the neutralization of edematogenic and nociceptive activities. In conclusion, geographic distribution influences the composition and activities of Bothriechis schlegelii venoms. Bothrops antivenom cross-reacted with these venoms and was partially effective in neutralizing some toxic activities of BsCo and BsCR.

Functional Mining of the Crotalus Spp. Venom Protease Repertoire Reveals Potential for Chronic Wound Therapeutics

“…Contrary to what is described above, our experimentation failed to demonstrate a differential protease activity on gelatin and casein zymographies, observing a limited number of bands on casein zymography, in the case of C. m. nigrescens, C. scutulatus, and C. atrox venoms. The band diminishing on casein gel is also described for other snake venoms such as Bothriechis schlegelii [80], Bothrops insularis [81], and Pseudoboa neuwieddi [82].…”

Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins

“…In order to study snake venom cytotoxicity, a variety of assays have previously been applied, ranging from in vivo wound exudate analysis by proteomics, immunochemical detection of ECM proteins in skin and exudates and various cell-based cytotoxicity assays to in vitro low-throughput assays such as zymography (utilizing non-fluorescent gelatin/collagen) and fluorometric assays assessing proteolytic activity with different fluorescently labelled peptide substrates (e.g., casein, rhodamine-110-peptide, desired peptide sequences) and self-quenched gelatin to assess protease activity [17,20,22,[27][28][29][30][31][32][33][34][35][36]. These assays vary considerably in model complexity, specificity, throughput and costs.…”

Protease Activity Profiling of Snake Venoms Using High-Throughput Peptide Screening

“…The results of the high-throughput peptide screening analysis not only provide a general overview of the magnitude of proteinase activity for each snake venom, but also provide insights into relevant targets for the enzymes involved in the pathophysiology of snakebite envenomings. The high-throughput method was effective in investigating multiple candidate substrates of proteinases, instead of a single or limited number that can be tested by traditional spectrophotometric, SDS-PAGE, or zymography techniques [19,20,31,32,33]. Typically, the thorough characterization of the proteinase substrate requires a combination of electrophoretic techniques such as SDS-PAGE or 2D-DIGE, and mass spectrometry methodologies (LC-MS/MS, MALDI-TOF) [34,35,36,37,38], although recently developed MS techniques can be used without the need for an electrophoretic step [39,40,41].…”

“…Snake venom proteinase activity has traditionally been investigated by the purification of a selected enzyme, followed by biochemical characterization. In these studies, the enzymatic activity of proteinases is examined in assays using general protein substrates, such as azocasein [19], by zymography [20], or with known targets of these enzymes, such as fibrinogenolytic, procoagulant, and platelet aggregation assays [12,14,21,22]. Recent studies have focused on the substrate specificity of proteinases of interest by the proteomic identification of cleavage sites.…”