Dq gelatin

The venom metalloprotease activity was analyzed using DQ-gelatin (ThermoFisher Scientific, UK) as a fluorogenic substrate. Various concentrations of Russell's viper venom (Kentucky Reptile Zoo, United States) were mixed with 10 μg/mL DQ-gelatin in a total reaction volume of 100 μL (final volume was made up using PBS) in a 96-well microtiter plate. The plate was incubated at 37 °C, and the level of fluorescence was measured at various time points using an excitation wavelength of 485 nm and emission at 520 nm in a Fluostar Optima (BMG Labtech, Germany) spectrofluorometer. Similarly, the serine protease activity was measured using Nα-benzoyl-
l-arginine 7-amido-4-methyl coumarin HCl (Sigma Aldrich, UK) as a fluorogenic substrate. Following the mixing of the substrate (2 μM) with different concentrations of Russell's viper venom, the plate was incubated at 37 °C and the resulting fluorescence was measured at an excitation wavelength of 366 nm and emission wavelength of 460 nm at various time points. The PLA
₂activity of Russell's viper venom was measured using an EnzCheck Phospholipase A
₂kit (ThermoFisher Scientific; dioleoyl phosphatidylcholine and dioleoyl phosphatidylglycerol as substrates) according to the manufacturer's instructions.

The protease activity assays were performed by using the EnzChek Elastase Assay Kit and EnzChek Gelatinase/Collagenase Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 10 µL of each bacterial supernatant (12 S. epidermidis (Table 1) and ATCC 12228) cultivated in 2 mL medium for 24h at 37°C, were incubated with 1 µg of DQelastin or DQgelatin (Thermo Fisher Scientific) diluted in the supplied reaction buffer (qsp 200 µL) in 96-well black plates (Corning, NY) for 20 hours. Relative fluorescent intensity was analyzed with a Fluorometer Infinite^® M200 PRO (Tecan system; excitation wavelength, 485 nm; emission wavelength, 538 nm).

The metalloprotease activity of both C. atrox whole venom and the purified protein was assessed using a fluorogenic substrate, DQ-gelatin (ThermoFisher Scientific, UK). Briefly, the whole venom or purified protein (10μg/mL) was mixed in phosphate buffered saline (PBS, pH 7.4) with DQ gelatin (10μg/mL). The reaction mix was incubated at 37°C and the level of fluorescence was measured at 60 minutes using an excitation wavelength of 485nm and emission wavelength of 520nm by spectrofluorimetry (FLUOstar OPTIMA, Germany).
Similarly, the serine protease activity was measured using a selective substrate, Nα-Benzoyl-L-Arginine-7-Amido-4-methylcoumarin hydrochloride (BAAMC) (Sigma Aldrich, UK). The whole venom or the purified protein (10μg/mL) was incubated with BAAMC (2μM) at 37°C and the level of fluorescence was measured at an excitation wavelength of 380nm and emission wavelength of 440nm by spectrofluorimetry.

The metalloprotease (collagenolytic) activity of the venom or the purified protein (CAMP-2) was measured using a fluorogenic substrate, DQ™-gelatin (ThermoScientific, Loughborough, UK). Several concentrations of the purified protein or venom (with and without different concentrations of batimastat, marimastat, EDTA, ZnCl₂ and CaCl₂ (Sigma Aldrich, Dorset, UK)) were added to a black 96-well plate in triplicates along with appropriate controls. Then DQ-gelatin (10 μg/mL) was added to each well, and following mixing, the plate was incubated at 37 °C and the level of fluorescence was measured at various time points using an excitation of 485 nm and emission wavelength of 520 nm in a FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany) spectrofluorimeter. To determine PLA₂ activity, an EnzChek™ Phospholipase A₂ Assay Kit (ThermoFisher Scientific, Loughborough, UK) was used in accordance with the manufacturer’s instructions.