*5.5. Inhibitory Assays*

Papain was utilized to select the chromatographic fractions that contained Cystatin-Hv, and, after obtaining the inhibitor in its homogeneous form, cathepsin L was used to determine the value of the inhibition constant (Ki). The assays were implemented according to Portaro et al. (2000) [46] with some minor modifications. Enzymes were preactivated for 15 min at room temperature with 6 mM DTT in 50 mM sodium phosphate, 200 mM NaCl, 5 mM EDTA, and pH 5.5 (final volume 100 μL). For the pool selection steps, 10 ng of papain, 5 μM of fluorogenic substrate Z-FR-AMC (Sigma-Aldrich, St. Louis, MO, USA) and 250 ng of protein from pooled purification fractions were used. Cathepsin L (0.4 nM) was

employed against three concentrations of purified Cystatin-Hv (8 nM, 16 nM and 24 nM) and two concentrations of the fluorogenic substrate Z-FR-AMC (1 Km and 2 Km, where the Km = 2.6 μM, ref. [47]) to determine the Ki value [48]. Control reactions were carried out in the same conditions but without Cystatin-Hv. The activity was measured (fluorescence at λEM 480 nm and λEX 360 nm) in a Victor 3 (Perkin Elmer, Boston, MA, USA) plate reader. The temperature remained constant at 37 ◦C, and one reading per minute was performed for 15 min, the plates being shaken before each measurement. The residual activity of human cathepsin L in the presence of Cystatin-Hv in different amounts was determined, and the inhibition constant (Ki) of Cystatin-Hv towards human cathepsin L was determined by the Dixon Plot equation (1/V vs. [I]) [49], using the software GraphPad Prism 5.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/toxins13120857/s1, Figure S1: Amino acid sequence of the mature Hviz340 recombinant protein and the tryptic peptides identified by LC-MS/MS analysis using a QExactive plus mass spectrometer and database search in PEAKS Studio X.

**Author Contributions:** Conceptualization, D.d.C.L., F.F. and A.M.C.-T.; methodology, D.d.C.L., F.F., R.T.K., F.C.V.P. and K.F.F.; investigation, D.d.C.L. and R.T.K.; formal analysis, D.d.C.L., R.T.K., A.M.X.P.A. and D.T.-S.; writing—original draft preparation, D.d.C.L., F.F. and F.C.V.P.; writing review and editing, D.d.C.L., F.F., R.T.K., F.C.V.P., A.M.X.P.A., D.T.-S. and A.M.C.-T.; supervision, F.F. and A.M.C.-T.; funding acquisition, D.d.C.L., F.F. and A.M.C.-T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Support Foundation for the Technological Research Institute: N◦500115A/PTC1043/15; São Paulo Research Foundation (FAPESP): #2013/07467-1, #2015/13124-5 and #2019/20832-7; FAPESP and GlaxoSmithKline: #2016/06026-0 and #2015/50040-4.

**Acknowledgments:** Authors acknowledges Tatiana Joly Siquini for the assistance with culture procedures and Viviane Barbosa Portas and Melissa Regina Fessel for the assistance with protein purification techniques and equipment handling.

**Conflicts of Interest:** The authors declare no conflict of interest.
