Oxiselect superoxide dismutase activity assay

SOD activity was measured using OxiSelect Superoxide Dismutase Activity Assay (Cellbiolabs, USA; STA-340) according to the manufacturer's protocol. Stationary phase L. infantum promastigotes (10⁹) were treated or not with 3 μM menadione (Sigma) O/N, washed twice in PBS and harvested by centrifugation at 6000 g for 5 min. The pellets were resuspended in 100 μl of cold 1 × Lysis Buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, protease inhibitor). Cells were lysed by three freeze–thaw cycles alternating liquid nitrogen and a 37 °C bath. The lysates were centrifuged at 15 000 g for 10 min at 4 °C, and protein contents were determined using a BCA protein assay system. The SOD assay system utilizes the xanthine oxidase, which oxidizes its substrate xanthine to produce superoxide anions. The included chromagen produces a water-soluble formazan dye upon reduction by superoxide anions. Inhibition of yellow formazan after the addition of whole-cell extracts to the SOD assay mix was determined as a measure of SOD activity. One unit of SOD will inhibit by 50% the rate of reduction of chromagen.

The algal culture treated with bacterial cells and cell free suspensions were collected every 24 h and tested by an antioxidative enzyme assay. For this, same amount (w) of cell pellet from control and each treatment were collected by transferring cultures to 2-mL sterilized tubes and centrifuged at 5,000 × g for 5 min. Supernatants were discarded and pellets were washed with PBS buffer three times. The washed cells were homogenized with ultrasonic cell pulverizer (Powersonic 603; Hwashin Technology Company) at 30°C for 30 min. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were measured using OxiSelect Superoxide Dismutase Activity Assay (Cell Biolabs Inc., USA), CAT100 (Sigma-Aldrich, USA), and OxiSelect Hydrogen Peroxide/Peroxidase Assay Kit (Colorimetric) (Cell Biolabs Inc., USA), respectively. All of the analytical methods followed the kit’s operation manual and absorbance values at different wavelengths were taken using a TECAN Spark 10M spectrophotometer (Männedorf, Zurich, Switzerland).