Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins
Eric Wachtel,
Matyas A. Bittenbinder,
Bas van de Velde,
Julien Slagboom,
Axel de Monts de Savasse,
Luis L. Alonso,
Nicholas R. Casewell,
Freek J. Vonk,
Jeroen Kool
Affiliations
Eric Wachtel
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Matyas A. Bittenbinder
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Bas van de Velde
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Julien Slagboom
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Axel de Monts de Savasse
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Luis L. Alonso
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Nicholas R. Casewell
Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
Freek J. Vonk
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Jeroen Kool
AIMMS, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM–polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms.
