Xanthine Oxidase

For enzyme extracts and assays, fresh roots (0.1 g) were ground in liquid nitrogen, and then suspended in 0.9 mL solution containing 10 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 4°C, 2500 rpm for 10 min and the resulting supernatant was collected for determination of the activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and glutathione peroxidase (GSH-Px, EC 1.11.1.9) using commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All enzymes above were detected using a microplate reader (SpectraMax M5, USA), and 5 to 10 seedlings were used to provide enough amounts of root tissues in each experimental replicate (n = 3).
The activity of SOD was determined by measuring the inhibiting rate of the enzyme to O₂⁻· produced by the xanthine morpholine with xanthine oxidase using the SOD assay kit. Each endpoint assay was detected the red substances of the reaction system by absorbance at 550 nm after 40 min of reaction time at 37°C. And one unit SOD activity (U) was defined as the quantity of SOD required to produce 50% inhibition of reduction of nitrite in 1 mL reaction solution by measuring the change of absorbance at 550 nm.
The CAT activity was measured based on the hydrolysis reaction of hydrogen peroxide (H₂O₂) with CAT, which could be terminated by molybdenum acid (MA) to produce yellow MA-H₂O₂ complex. CAT activity was calculated by the decrease in absorbance at 405 nm due to the degradation of H₂O₂, and one unit is defined as the amount of enzyme that will cause the decompose of 1 µmol hydrogen peroxide (H₂O₂) per second at 37°C in 1.0 g fresh tissue according to CAT assay kit.
The POD activity was measured based on the change of absorbance at 420 nm by catalyzing H₂O₂. One unit was defined as the amount of enzyme which was catalyzed and generated 1 µg substrate by 1.0 g fresh tissues in the reaction system at 37°C. POD activity was calculated as the formula according to POD assay kit.
The GSH-Px activity was also measured using the assay kit based on the principle that oxidation of glutathione (GSH) and hydrogen peroxide (H₂O₂) could be catalyzed by GSH-Px to produce oxidized glutathione (GSSG) and H₂O. In addition GSH reacts with 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) to produce stable yellow substances and the decrease of GSH at 412 nm during the reaction is indicative of GSH-Px activity in tissues. One GSH-Px unit of GSH-Px activity (U) was calculated as the amounts of enzyme that will oxidize 1 µmol/L GSH in reaction system at 37°C per minute in 1.0 g fresh tissue according to the assay kit. All of the enzymes were expressed as in U/g FW.

The concentration of malondialdehyde (MDA) as a marker of oxidative stress and the levels of total antioxidant capacity (TAC), catalase (CAT) and superoxide dismutase (SOD) activity were measured using commercial kits and according to the manufacturer’s protocol (Kiazist, Iran). Briefly, kidney tissues were homogenized in lysis buffer containing protease inhibitors (Sigma–Aldrich, USA). After centrifugation by a 3-18KS Sigma centrifuge (Sigma, Germany), supernatants were collected for next analysis. MDA level was quantified by measuring thiobarbituric acid reactive substances produced in the reaction of MDA with thiobarbituric acid. TAC level was measured based on the capacity to convert Cu²⁺ to Cu⁺ ion. The activity of catalase was determined according to the reaction of the enzyme with methanol in the presence of hydrogen peroxide and measurement of generated formaldehyde. SOD activity was assayed by measuring the dismutation of superoxide radicals generated by the xanthine/xanthine oxidase system. Protein concentration of lysates was measured using Bradford method. Then the levels of oxidative stress markers were normalized to protein content [20 (link), 21 (link)].

Based on the metabolomics results, three key enzymes involved in essential pathways, including xanthine oxidase (XOD), pyruvate kinase (PK), and glucose 6-phosphate dehydrogenase (G6PDH), were examined for changes of their activities under desiccation stress. In brief, Salmonella cell suspensions at different sampling points were diluted with sodium phosphate buffer (0.5 M, pH 7.0) to obtain a final concentration of 10⁷ CFU/mL. The extraction solution was added according to the manufacturer’s instructions (Comin Biotechnology Co. Ltd., Suzhou, China). The mixture was ultrasonically broken with an ultrasonic ice bath (200 W, ultrasonic time 3 s at intervals of 10 s, and 30 times of ultrasound treatment). The supernatant was obtained by centrifugation at 8,000 × g and 4°C for 10 min and then incubated at room temperature for 30 min after adding the reaction reagent. UV spectrophotometric (UV–VIS, Thermo Fisher Scientific, United States) analysis was used to measure the concentration change of the reaction products catalyzed by specific enzymes. The characteristic absorption wavelength was 290 nm for XOD, and 340 nm for PK and G6PDH, respectively. The enzyme activity was determined in units of nmol/min/10⁴ cells.
In addition, the effect of continuous stress on the intracellular ATP content was also determined to further verify the regulation of metabolic pathways. Salmonella cell suspensions at different sampling points were centrifuged at 5,000 rpm for 5 min at 4°C, and then the supernatant was removed. Five milliliter of phosphate buffer saline solution (PBS, pH7.2) were added for resuspension. Cell suspensions were broken with ultrasound in an ice bath for 5 min. The supernatants were collected after centrifugation at 12,000 rpm for 20 min at 4°C. The ATP levels were assayed using an ATP assay kit (Jiancheng Bioengineering Institute, Nanjing, China). The results were analyzed by UV–VIS with an absorption wavelength of 636 nm. All assays were performed in biological triplicates.
The results were expressed as mean ± standard deviation. ANOVA was performed in SPSS 22.0 to determine significant differences between groups (p < 0.05).

SUA, serum creatinine (CRE), and blood urea nitrogen (BUN) concentrations were determined with commercial kits from the Jiancheng Biotech (Nanjing, China), and Xanthine oxidase (XOD) and adenosine deaminase (ADA) activities in livers were determined with commercial kits form the Jiancheng Biotech (Nanjing, China), following the manufacturer’s instructions.