Ferrozine

All assays were made using 96-well microplates (Nunclon, Nunc, Roskilde, Denmark) and were measured in an ELISA Reader Infinite Pro 200F (Tecan Group Ltd., Männedorf, Switzerland).
The DPPH^• (2,2-diphenyl-1-picryl-hydrazyl) radical scavenging activity was measured by a previously described method [57 (link)]. DPPH solution (180 μL of freshly prepared 0.07 mg/mL solution) was mixed with 20 μL of the examined extract in various concentrations in microplates. The absorbance at 517 nm was monitored after 30 min incubation at 28 °C and the results were expressed as an EC₅₀ value. Ascorbic acid was used as the control. The antiradical potential was also analyzed using the previously described ABTS^•+ (2,2′-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid) assay [57 (link)]. The absorbance was measured at 734 nm and the results were expressed as millimoles of Trolox equivalents per g of dry extract (TEAC).
The metal chelating activity was determined by the method described by Guo et al. [58 ] with some modifications. In this assay, 0.2 mM aqueous solution of ferric chloride and 0.5 mM aqueous solution of ferrozine were used. Twenty microliters of the 0.2 mM aqueous solution of ferric chloride (II) was mixed with 100 μL of extract at different concentrations. Next, 40 μL of 0.5 mM aqueous solution of ferrozine was added and microplates were shaken and incubated for 10 min in 24 °C. The absorbance was measured at 562 nm and the percentage of inhibition of ferrozine–Fe²⁺ complex formation was calculated using the following formula:
where A_c is the absorbance of the control (water instead of the extract), and A_s is the absorbance of the extract.
The results were presented as the concentration of the extract that causes metal chelating in 50% (EC₅₀) calculated on the basis of the linear correlation between the inhibition of ferrozine–Fe²⁺ complex formation and the concentrations of the extract. EDTA was used as a positive control.
Antioxidant activity was also assayed using the β-carotene bleaching method described and modified by Deba and co-authors [51 (link)]. Twenty microliters of extract at different concentrations were mixed with freshly prepared β-carotene-linoleic acid emulsion, and incubated for 20 min at 40 °C. The absorbance was measured at 470 nm. BHT was used as a positive control.

The chelating effect on ferrous ions of the prepared extracts was estimated by the method of Dinis with slight modifications [10 (link)]. Briefly, 100 μL of each test sample (1 mg/mL) was taken and raised to 3 mL with methanol. 740 μL of methanol was added to 20 μL of 2 mM FeCl₂. The reaction was initiated by the addition of 40 μL of 5 mM ferrozine into the mixture, which was then left at room temperature for 10 min and then the absorbance of the mixture was determined at 562 nm.
(i) Ferric Ion Reducing Antioxidant Power (FRAP Assay). FRAP activity was measured according to the method of Benzie and Strain [11 (link)]. Briefly, acetate buffer (300 mM, pH 3.6), TPTZ (2,4,6-tripyridyl-s-triazine) 10 mM in 40 mM HCl and FeCl₃·6H₂O (20 mM) were mixed in the ratio of 10 : 1 : 1 to obtain the working FRAP reagent. Test sample (0.5 mL) was mixed with 3 mL of working FRAP reagent and absorbance was measured at 593 nm after vortexing. Methanol solutions of FeSO₄·7H₂O ranging from 100 to 2000 μM were prepared and used for the preparation of the calibration curve of known Fe²⁺ concentration. The parameter equivalent concentration was defined as the concentration of antioxidant having a Ferric-TPTZ reducing ability equivalent to that of 1 mM FeSO₄·7H₂O.

CD activity assay was conducted as previously described (Siegel, 1965 (link); Yang et al., 2015 (link); Li et al., 2018 (link)). Briefly, purified recombinant CDs (5 μM) were incubated with buffer A in the presence of 3 mM dithiothreitol (DTT) for 5 min at 37°C. L-cysteine (2 mM) was added to initiate the CD activity reaction. Reactions were terminated by the addition of 20 mM N, N-dimethyl-p-phenylene-diamine sulphate (in 7.2 M HCl), and 30 mM FeCl₃ (in 1.2 M HCl). The colour was left to develop for 20 min at 37°C before quantifying methylene blue at 669 nm. Buffer A was used as the negative control and endonuclease III (Nth) was used as the positive control. The iron content in E. coli Nth was calculated from the iron-ferrozine determination, as previously described by Ren et al. (2021) (link), and because E. coli Nth contains a stable [4Fe-4S] cluster, the protein should have equal amounts of iron and sulphur. Therefore, the extinction coefficient of sulphur was calculated based on the content of iron in Nth. Spectra were recorded every 5 min for 15 min.

Strains were grown to mid-exponential phase (OD₆₀₀ = ~0.4 to 0.6) in chemically defined synthetic medium or chemically defined synthetic medium supplemented with 0.5 mM flavin adenine dinucleotide (FAD) and then washed twice with 1× phosphate-buffered saline (PBS). Washed bacteria were resuspended in chemically defined synthetic medium supplemented with 4 mM Ferrozine and normalized to an OD₆₀₀ of 0.5. To conduct the assay, 100 μL of resuspended bacteria was mixed with 100 μL of chemically defined synthetic medium supplemented with 100 mM ferric ammonium citrate and transferred into a 96-well plate in triplicate. Measurements were done using a plate reader with the temperature set at 37°C, and the absorbance was read at 560 nm every 30 s for 1.5 h. Maximal rates (typically over 2 min) were calculated and reported as a percentage of the ferric iron reductase activity of the wild type.

Hematology was analyzed using an automated machine (BC-5000Vet, MINDRAY, Shenzhen, PR China). The blood biochemistry (BUN, CR, TP, ALB, and Pi) was analyzed using an automated machine (ILAB 650 Chemistry Analyzer, Diamond diagnostics, Holliston, MA, USA). Serum and urine iron concentration and serum UIBC were analyzed by the standard colorimetric ferrozine method (Cobas c501, Roche Diagnostics, Indianapolis, IN, USA). The TIBC was calculated by the sum of plasma iron and UIBC. The TF level in both plasma and urine was analyzed by enzyme-linked immunosorbent assay method (Canine TF ELISA Kit [ab157704], Abcam, Cambridge, UK). The urinary protein was analyzed by multicolor method (Olympus Au 400, Olympus America Inc., Melville, NY, USA). Urinary protein, iron, and TF were divided by urinary CR and expressed as UPC ratio, urinary iron per CR ratio (U-Iron/CR), and urinary TF per CR ratio (U-TF/CR), respectively.