*2.2. Stability of Hypotensins against Metallopeptidases ACE and NEP*

As hypotensins showed interactions, even if opposite, in relation to the studied vasopeptidases, tests to determine the susceptibility to hydrolysis of both synthetic peptides were carried out. For this, the hypotensins were incubated with the enzymes, in their respective buffers, and later analyzed in an HPLC-C18 system.

Both hypotensins were resistant to hydrolysis and, therefore, did not behave as substrates for ACE and NEP, even after 4 h of incubation at 37 ◦C (Figure 1).

**Figure 1.** Analysis of the susceptibility to hydrolysis of the hypotensins over the vasopeptidases NEP and ACE. Incubations were carried out in Tris HCl 100 mM, NaCl 50 mM and ZnCl2 10 μM, pH 7.0, for the ACE assays, and, for the NEP assays, in Tris HCl 50 mM, pH 7.5 buffer, for four hours at 37 ◦C. (**A**) TsHpt-I control (black line) and after incubation with NEP (red line). (**B**) TsHpt-II control (black line) and TsHpt-II incubated with NEP (red line). (**C**) TsHpt-I control (black line) and incubated with ACE (red line). (**D**) TsHpt-II control (black line) and after incubation with ACE (red line). The samples of negative and experimental controls (**A**) TsHpt-I and (**B**) TsHpt-II are shown on the left and right, respectively. The hydrolysis products were considered ≤5%. The gradient used was 20% to 60% solvent B in 20 min, and 60% to 100% solvent B in 5 min (flow 1 mL/min, absorbance at 214 nm). Restek Ultra C-18 5 μm column, 250 × 4.6 mm.

Since TsHpt-I demonstrated inhibition of NEP catalytic activity, and was resistant to hydrolysis, kinetic analyses to determine the inhibition constant (Ki) and the mechanism of inhibition of NEP by TsHpt-I were carried out.

TsHpt-I behaved as a non-competitive inhibitor of NEP, presenting an inhibition constant (Ki) of 4.35 μM (Figure 2). The determination of the mechanism and Ki of TsHpt-II on neprilysin activity were not performed as this peptide showed weak interaction with the peptidase (Table 1).

**Figure 2.** Lineweaver–Burk plot of NEP inhibition by TsHpt-I. The inhibition constant (Ki) was determined using four Abz-RGFK-EDDnp substrate concentrations (4 μM, 6 μM, 8 μM and 10 μM), with variation in the concentration of TsHpt-I (3 μM and 4 μM), keeping the amount of enzyme fixed (1.5 ng). The experiment was performed in triplicate.

TsHpt-I is the second neprilysin inhibitor from the *Tityus serrulatus* venom described in the literature. Previous observations by our group revealed the presence of a peptide capable of inhibiting human NEP and an NEP-like present in cockroaches, the [des-Arg1]- Proctolin [16]. [des-Arg1]-Proctolin was characterized as a competitive inhibitor of NEP, with an inhibition constant of 0.94 μM [16]. Thus, there may be a synergistic action between these two peptides present in TsV, TsHpt-I and [des-Arg1]-Proctolin, for the inhibition of NEP, and this fact may be related to hypotension associated with scorpionism.
