*5.3. Stability Test of the Synthetic Peptides*

The synthetic peptides TsHpt-I and TsHpt-II (30 μM) were incubated with ACE (3.0 ng), in Tris HCl 100 mM, NaCl 50 mM and ZnCl2 10 μM, pH 7.0 buffer, and NEP (1.5 ng), in Tris HCl 50 mM, pH 7.5 buffer, at 37 ◦C for 4 h. Samples containing only the synthetic peptides were used as the negative control. After incubation, samples were analyzed by reverse phase chromatography on RP-HPLC (Shimadzu, Kyoto, Japan), using a Restek Ultra C-18 column (5 μm, 250 × 4.6 mm). Solvents used were 0.1% TFA in water (solvent A), and acetonitrile plus solvent A (9:1) as solvent B. Separations were performed at a flow rate of 1 mL/min and a 20–60% gradient of solvent B over 20 min. In all cases, elution was followed by the measurement of ultraviolet absorption (214 nm).

#### *5.4. Characterization of TsHpt-I as a NEP Inhibitor*

To determine the inhibition constant (Ki) of TsHpt-I over NEP, four concentrations of Abz-RGFK (Dnp)-OH (4 μM, 6 μM, 8 μM and 10 μM) and 3 μM and 4 μM of TsHpt-I were tested using 1.5 ng of peptidase in 100 μL of final volume of Tris HCl pH 7.5. The Km value of the substrate used was determined to be 14 μM [27]. Controls without the TsHpt-I were also performed in all assays. The reactions were monitored as described above (item 5.2). The Lineweaver–Burk plot was constructed (1/V × 1/(S)) according to the presented mechanism. The Ki was calculated as described by Segel [28]. All assays were performed in triplicate.
