5.3.1. Enzymatic Assays on Synthetic Substrates

SVMP, SVSP, and PLA2 enzymatic activities of individual venoms from *B. atrox* were assayed using synthetic substrates according to the procedures previously standardized in our lab [42]. Briefly, the substrate used for SVMPs was Abz-AGLA-EDDnp substrate (Peptide International, Gardner, MA, USA), and the results are expressed as Relative Fluorescence Units-RFU/min/μg. The PLA2 activity was assayed using the substrate 4-nitro-3-[octanoyloxy] benzoic acid (Enzo Life Sciences, New York, NY, USA) and activity were determined according to the absorbance at 425 nm and expressed as Absorbance/min/μg of venom. For SVSP catalytic activity, the chromogenic synthetic substrate benzoyl-arginylp-nitroanilide (L-BAPNA) (Sigma-Aldrich®, St. Louis, MO, USA) was used and the hydrolysis of the substrate was expressed as the increase in Absorbance/min/μg of venom. The results represent the mean ± SD of three independent experiments, undertaken in triplicate.
