The activity of recombinant human ACE2 is also measured by its ability to cleave a fluorogenic peptide substrate MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-OH in the assay buffer 50 mM Tris, 1 M NaCl, pH 7.5. The rhACE2 is diluted to 0.5 ug/mL in assay buffer. Loading into a black well plate 50 μL of 0.5 ug/mL rhACE2 and start the reaction by adding 50 μL of 20 μM substrate, with a substrate blank containing 50 μL assay buffer, 50 μL substrate, and no rhACE2. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The specific activity of recombinant human ACE2 is > 1100 pmol/min/μg.

Angiotensin-converting enzyme 2 (ACE2), as a transmembrane protein, serves as the main entry
point into cells for some coronaviruses, including HCoV-NL63, SARS-CoV , and SARS-CoV-2 . More specifically, the binding of the spike S1 protein of SARS-CoV and SARS-CoV-2 to the enzymatic domain of ACE2 on the surface of cells results in endocytosis and translocation of both the virusand the enzyme into endosomes located within cells. Besides, recent studies show that spike (S) proteins of 2019-nCoV and SARS-CoV may use the same host cell receptor called angiotensin-converting enzyme 2 (ACE2) for entering into host cells, thus a binding ELISA assay was conducted to detect the interaction of recombinant human ACE2 and recombinant Spike glycoprotein RBD. Briefly, biotin-linked ACE2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to RBD-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST 3 times and incubation with HRP conjugage for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of ACE2 and RBD was shown in Figure 1, and this effect was in a dose dependent manner.