*2.11. Analysis of Enzymatic Activities*

Promastigotes (5 × 106 protozoa/mL) were treated with 1/5 IC50/4 h NaNO2 in HBSS at 25 ◦C for 4 h, washed with PBS, and kept dry at −20 ◦C until use to ensure that all biological replicas were extracted and analyzed at the same time and under the same experimental conditions. Protein homogenates were prepared by sonication, as previously described [32]. Briefly, pellet was resuspended in cold PBS containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and disrupted on ice by sonication for 10 cycles of 7 s with intervals of 7 s, using a Markson GE50 Ultrasonic Processor. Amplitude was set to 70%, and parasite disruption was monitored by light microscopy. Soluble fraction was obtained, and protein concentration was measured using Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Palo Alto, CA, USA). For glutathione peroxidase (GPx) assay, soluble protein extract (0.5 mg/mL) was incubated in 100 mM KH2PO4 (pH 7.8) buffer, supplemented with 1 mM reduced glutathione (Sigma-Aldrich, St. Louis, MO, USA), 5 U/mL glutathione reductase (Sigma-Aldrich, St. Louis, MO, USA), 200 μM NADPH (Sigma-Aldrich, St. Louis, MO, USA), and 300 μM H2O2. The rate of the NADP (ε = 6.22 M<sup>−</sup>1cm−1) reduction was determined at 340 nm. Lactate dehydrogenase activity was evaluated using a commercial kit (Doles, Goiânia, Brazil) following the manufacturer's protocol, with some modifications. Briefly, 0.5 mg/mL of protein was added to 100 μL manufacturer's substrate solution and 10 μL ferric alum for 2 min at 37 ◦C. After that, 10 μL NAD+ + phenazine methosulfate (PMS) was added, and absorbance measured for 45 min (every 1 min) at 510 nm. A standard curve was generated with lactate dehydrogenase (Sigma-Aldrich, St. Louis, MO, USA). All enzyme activities were measured at 37 ◦C in a total reaction volume of 200 μL using a SpectraMax Plus384 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
