Quantitative ACE2 Enzymatic Activity Assay

ACE2 enzymatic activity assay was performed as previously published by our group [25 (link),44 (link),45 (link),46 (link)] and adapted to different tissues. Briefly, 5 μL of serum or 5 μg of tissue samples that were previously homogenized were incubated using a 100 mM Tris-HCl, 600 mM NaCl, 10 μM ZnCl₂, pH 7.5 buffer in the presence of protease inhibitors containing 100 μM captopril, 5 μM amastatin, 5 μM bestatin (all from Sigma-Aldrich, Madrid, Spain), and 10 μM Z-Pro-prolinal (Enzo Life Sciences, Grupo Taper, Madrid, Spain). Samples were incubated with 20 μM Mca-Ala-Pro-Lys(Dnp)-OH (Enzo Life Sciences), a specific ACE2 quenched fluorogenic substrate, at 37 °C. Enzymatic activity was determined after 4 hours of incubation in tissue, and 16 h of incubation in serum. The plates were read using a fluorescence plate reader Tecan Infinite 200 (TECAN Instruments) at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Results were expressed as RFU (Relative Fluorescent Units) per μL of sample or μg of protein and per hour (RFU/μl/h or RFU/μg/h).