*2.5. Analysis of rFhSOD1 and rFhSOD3 Enzyme Activity*

The enzymatic activity of the recombinant FhSODs was measured via the adaptation of an existing xanthine oxidase (XOD)-based SOD assay, whereby O2 •- is enzymatically produced because of the conversion of xanthine to H2O2 and uric acid, which in turn transforms nitroblue tetrazolium (NBT) to NBT-diformazan dye [27]. To determine the

activity of our recombinant enzymes under physiological conditions compatible with the parasite, our assay was conducted at 37 ◦C in 200 μL assay buffer (1 × PBS; Sigma-Aldrich, 0.1 mM Hypoxanthine; Sigma-Aldrich, 0.1 mM DTPA; Sigma-Aldrich, 2.5 μL/mL tetrazolium salt; Cayman Chemical, Ann Arbor, MI, USA). All other enzymes (native XOD from bovine milk; Roche, used at a final concentration of 8.0 × <sup>10</sup>−<sup>3</sup> U/mL), including the standard curve (native bovine erythrocyte SOD, BS; Sigma-Aldrich), were diluted in PBS prior to use. Recombinant proteins and the standard curve (BS) were serially diluted 1:1 in PBS (rFhSOD1 and rFhSOD3: 10–0.3125 μg/mL; BS: 100–3.125 μg/mL, corresponding to 4–0.125 U/mL) and run in duplicate at a final assay volume of 250 μL. The assay was read continuously for 30 min at 450 nm using a microplate reader (PolarStar Omega Spectrophotometer; BMG LabTech, Ortenburg, Germany) immediately after the addition of XOD.
