*5.2. Interactions of Hypotensins with Vasopeptidases ACE and NEP*

For ACE assays, 10 μM of each peptide was incubated with 3.0 ng of peptidase and 10 μM of Abz-FRK (Dnp) P-OH substrate, in Tris HCl 100 mM, NaCl 50 mM and ZnCl2 10 μM, pH 7.0 buffer. For NEP, 10 μM of TsHpt-I or -II was incubated with 1.5 ng of peptidase, 3.5 μM Abz-RGFK (Dnp)-OH in Tris HCl 50 mM, pH 7.5 buffer. All reactions occurred at 37 ◦C, in a final volume of 100 μL, in a Victor 3 fluorimeter (Perkin–Elmer, Waltham, MA, USA) adjusted for excitation and emission readings at 320 and 420 nm, respectively, for 15 min (one reader per minute). Results were obtained in triplicate and analyzed using GraFit 5 (Erithacus software, East Grinstead, West Sussex, UK).
