4.6. Enzymatic Activity

The protocol designed by Prasa et al. [49] was used to perform the enzymatic assay with detection at a wavelength of 405 nm. The reagents used included 50 mM Tris-HCl buffer (pH 7.4), 100 mM NaCl, and 2 mg/mL Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA), a suitable trypsin substrate. Furthermore, three assays were done including the substrate only, 1 mg/mL protein incubated with the substrate, and 1 mg/mL gyroxin incubated with the substrate. The reagents were then pipetted onto a microplate reader incubated at 37 °C. The same protocol was followed for the determination of phospholipase A2 activity at a wavelength of 425 nm, using 50 mM Tris-HCl buffer (pH 7.8), 100 mM NaCl, and the 4-nitro-3-octanoyloxybenzoic (NOB) protein as a substrate (2 mg/mL). The readings were performed on a UV-VIS SpectraMax 190 (Molecular Devices, LLC., 3860 N First Street, San Jose, CA, USA).