Spectramax m2 fluorimeter

SVMPs, SVSPs and PLA2 enzymatic activities were assayed using synthetic substrates according to the procedures previously standardized in our lab [66 (link)]. For SVMPs, venom samples (10 $\mathsf{\mu }$ g) were incubated with 50 $\mathsf{\mu }$ M of Fluorescence Resonance Energy Transfer (FRET) Abz-AGLA-EDDnp substrate (GenOne Biotechnologies, Rio de Janeiro, RJ, Brazil), and the enzymatic reactions were monitored in a SpectraMax^® M2 fluorimeter (Molecular Devices, San Jose, CA, USA) with excitation at 340 nm and emission at 415 nm, at 37 ${}^{\circ }$ C in kinetic mode over 10 min with a read range of 1 min. The results were expressed in Relative Fluorescence Units-RFU/min/ $\mathsf{\mu }$ g. The PLA2 activity of venom samples (20 $\mathsf{\mu }$ g) was assayed using 500 $\mathsf{\mu }$ M of the substrate 4-nitro-3-[octanoyloxy] benzoic acid (Enzo Life Sciences, New York, NY, USA) incubated for 40 min at 37 ${}^{\circ }$ C and activity determined according to the absorbance at 425 nm and expressed as Absorbance/min/ $\mathsf{\mu }$ g of venom. For SVSPs, the venom was incubated using 800 $\mathsf{\mu }$ M of substrate N $\alpha$ -benzoyl-arginyl-p-nitroanide (L-BAPNA; Sigma-Aldrich, Darmstadt, Hesse, Germany) incubated for 40 min at 37 ${}^{\circ }$ C and according to absorbance at 405 nm and expressed as Absorbance/min/ $\mathsf{\mu }$ g. Differences in the enzymatic activity between both species were assessed using a One-Way ANOVA and considered statistically significant at p-value < 0.05.

Enzymatic activities on synthetic substrates of metalloproteinases (SVMPs) and serine proteinases (SVSPs) were evaluated according to the methods described by Knittel et al. (2016) [118 (link)]. For the assay with SVMPs, 10 μg of venom diluted in reaction buffer (50 mM Tris-HCl, 10 mM CaCl₂, 150 mM NaCl and 0.05% Brij 35-Sigma-Aldrich, pH 7.5) were incubated with 50 μM of FRET (fluorescence resonance energy transfer) substrate Abz-AGLA-EDDnp (Peptide International), and enzymatic reactions were monitored in a SpectraMax^® M2 fluorimeter (Molecular Devices, San Jose, CA, USA) with excitation at 340 nm and emission at 415 nm at 37 °C in kinetic mode. As a positive control, Bothrops jararaca venom (10 μg) was used. Results were expressed in relative fluorescence units (RFU/min/μg). The SVSPs activity of venom samples was determined using 800 μM Nα-benzoyl-arginyl-p-nitroanide substrate (L-BAPNA–Sigma Life Science) incubated with 30 μg of venom diluted in 10 mM Tris-HCl buffer, pH 7.5 for 40 min at 37 °C. As a positive control, we used Bothrops neuwiedi venom (30 μg). Activity was determined according to absorbance at 425 nm and expressed as absorbance/min/μg of venom.