4.2. Fluorogenic Reporter Assays

Ten fluorogenic peptide reporter substrates each containing a C-terminal reporter group 7-amino-4-methylcoumarin (AMC) were dissolved in DMSO at a concentration of 500 µM. An equal volume of five reporter substrates, namely Z-Arg-Arg-Leu-Arg-AMC (System Peptide Company, Shanghai, China), Boc-Ala-Gly-Pro-Arg-AMC (Enzymes Systems Products, Livermore, CA, USA), N-Benzoyl-Phe-Val-Arg-AMC (Sigma, Burlington, MA, USA), Boc-Leu-Arg-Arg-AMC (Boston Biochem, MA, USA) and Glutaryl-Gly-Arg-AMC (Bachem, Torrance, CA, USA), were combined to form Substrate Pool A. An equal volume of five additional reporter substrates, namely, Arg-AMC, Boc-Val-Arg-AMC, Z-Arg-Arg-AMC, Gly-Arg-AMC (all from Bachem, Torrance, CA, USA) and Z-Phe-Arg-AMC (R&D Systems, Minneapolis, MN, USA), were combined to form Substrate Pool B. Pool A and B were diluted 20-fold in Assay Buffer 1 (Dulbecco’s phosphate-buffered saline, 1 mM DTT, 0.01% Tween-20) such that the final concentration of each substrate was 5 µM. Fifteen microliters of the substrates were added to multiple wells in a black 384-well plate (Thermo Scientific, Waltham, MA, USA, Part # 262260) that contained 15 µL of plasma that was previously diluted 15-fold in Assay Buffer 1. The final composition of each well consisted of five fluorogenic reporter substrates each at 2.5 µM and pig plasma diluted 30-fold in Assay Buffer 1. Fluorescence was measured at 22 °C using a Synergy HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) with excitation and emission wavelengths of 360 nm and 460 nm, respectively. Protease activity was reported as the change in relative fluorescent units per second. The mean and standard deviation for the activity readings from the six pig plasma samples were calculated.