Determination of ACE2 Activity

The ACE2 activity assay was carried out according to a published protocol with minor modifications (Shao et al., 2013; Xiao and Burns, 2017). Briefly, 15 μL of each plasma sample was diluted with an enzyme buffer consisting of 1 M NaCl, 75 mM Tris-HCl, 5.0 mM ZnCl₂, pH 6.5 with protease inhibitors, which were 10 μM captopril, 5 μM amastatin, and 10 μM bestatin (all from Sigma Aldrich, St. Louis, Missouri, United States) and 10 μM Z-prolyl-prolinal (Enzo Life Sciences, Inc., NY, United States). The ACE2-specific quenched fluorescent substrate, MCA-Ala-Pro-Lys-2, 4 dinitrophenyl (AnaSpec, Inc., San Diego, CA, United States) diluted in enzyme buffer were added to the samples in a final concentration of 50 μM in 100 μL on a black 96-well microplate. The plate was covered with aluminum foil to protect from light and incubated at room temperature for 24 h on a plate shaker. We have used recombinant human ACE2 (R&D Systems, Minneapolis MN) as the standard instead of using a fluorescent product to measure ACE2 activity. ACE2 cleaves the Pro-Lys bond of the substrate, and the Relative Fluorescence Units (RFU) of the released Mca-Ala-Pro was measured over a period of 24 h. The plate was read on a fluorescence reader (Clario Star, BMG Labtech, GmbH, Ortenberg, Germany) with an excitation wavelength of 320 nm and emission wavelength of 405 nm. The specificity of the enzyme activity was checked using 1 μM of an ACE2 inhibitor (DX600; AnaSpec, Inc., San Diego, CA, United States), which is specific for human ACE2. We calculated how much ACE2 activity there was in a sample comparing the RFU to the RFU of known concentrations of recombinant ACE2, that were treated in an identical manner. Thus ACE2 activity is reported as the activity/ng/ml of ACE2. All samples were assayed in duplicate. The mean intra and inter-assay coefficients of variations were 7.17% (n = 136) and 4.16% (5 plates), respectively.