Tetrazolium salt

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 (link)]. 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 × 10⁻³ 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.

Left ventricles were homogenized in ice-cold buffer (50 μl per mg wet weight) consisting of 50 mM KH₂PO₄, 1 mM EDTA and 0.05% Triton X-100, pH 7.5. The homogenate was centrifuged at 1400 g for 1 min at 4°C. The supernatant was used for analyses of citrate synthase (CS), β-hydroxyacyl-coenzyme A dehydrogenase (β-HAD) and glutathione S-transferase (GST) activities using standard spectrophotometric techniques [30 (link), 31 (link)]. Activities were measured at room temperature under conditions that yield linearity with respect to extract volume and time. The protein content was determined using the Bradford assay and activities were adjusted for protein content. The total SOD activity was measured with a kit based on the reduction of tetrazolium salt induced by superoxide production (Cayman Chemicals) as described previously [32 (link)]. The mitochondrial ATP-production rate (MAPR) in isolated mitochondria was determined with a firefly luciferase method at 25°C by using a 1251 luminometer (BioOrbit Oy) as previously described [33 (link)]. MAPR was determined in the presence of palmitoyl-carnitine at four different concentrations and presented as the ATP synthesis rate (units) per unit of CS activity (MAPR/CS).