Milliq 50 glycerol

A working stock solution of freeze dried venom was reconstituted in a buffer containing 50% MilliQ/50% glycerol (>99.9%, Sigma, St. Louis, MO, USA) at a 1:1 ratio to preserve enzymatic activity and reduce enzyme degradation. Varying concentrations of crude venom (10 ng/µL and 50 ng/µL) were plated out in triplicates on a 384-well plate (black, Lot#1171125, nunc™ Thermo Scientific, Rochester, NY, USA) and measured by adding 90 µL quenched fluorescent substrate per well (total volume 100 µL/well, 10 µL/5mL enzyme buffer, 150 mM NaCl, and 50 mM Tri-HCl (pH 7.3), Fluorogenic Peptide Substrate, R&D systems, Cat#ES001 & ES011, Minneapolis, MI, USA). Fluorescence was monitored by a Fluoroskan Ascent™ (Thermo Scientific, Vantaa, Finland) Microplate Fluorometer (Cat#1506450, Thermo Scientific, Vantaa, Finland) (Cat#ES001 for Matrix Metalloprotease at an excitation of 320 nm, emission at 405 nm; Cat#ES011 for Kallikrein at an excitation of 390 nm, emission at 460 nm) over 400 mins or until activity had ceased. Data was collected using Ascent^® Software v2.6 (Thermo Scientific, Vantaa, Finland).

We assessed the continuous Phospholipase A₂ (PLA₂) activity of the venoms using a fluorescence substrate assay (EnzChek^® Phospholipase A₂ Assay Kit, Cat#E10217, Thermo Scientific, Rochester, NY, USA), measured on a Fluoroskan Ascent^® Microplate Fluorometer (Cat#1506450, Thermo Scientific, Vantaa, Finland). As above, we used a working stock solution of freeze dried venom reconstituted in a buffer containing 50% MilliQ/50% glycerol (>99.9%, Sigma) at a 1:1 ratio. A concentration of enzyme activity in venom (50 ng/µL) was brought up in 12.5 µL 1× PLA₂ reaction buffer (50 mM Tris-HCL, 100 mM NaCl, 1 mM CaCl₂, pH 8.9) and plated out in triplicates on a 384-well plate (black, Lot#1171125, nunc™ Thermo Scientific, Rochester, NY, USA). The triplicates were measured by dispensing 12.5 µL quenched 1 mM EnzChek^® (Thermo Scientific, Rochester, NY, USA) Phospholipase A₂ Substrate per well (total volume 25 µL/well) over 100 min or until activity had ceased (at an excitation of 485 nm, emission 538 nm). Purified PLA₂ from bee venom (1 U/mL) was used as a positive control and data was collected using Ascent^® Software v2.6 (Thermo Scientific, Vantaa, Finland).