Ferroxidase activity assays on Rhodospirillum rubrum, Pyrococcus furiosus, and Haliangium ochraceum Encapsulated ferritins

Ferritin ferroxidase centre (FOC) catalytically converts Fe(II) into less toxic Fe(III). Ferroxidase assays were conducted to detect formation of iron(III) species at 315 nm by UV-vis spectroscopy. Moreover, inhibition of the enzymatic reaction by addition of competitive Zn(II) was tested in the same way.Protein samples (10 uM) were prepared by diluting purified protein fractions in 10 mM Hepes pH 8.0, 150 mM NaCl under anaerobic conditions. Oxygen-free aliquots of 1mM Fe(II) and Zn(II) were prepared by anaerobically dissolving FeSO4.7H2O or ZnSO4 in 0.1% (v/v) HCl, respectively.Metal and protein samples were then added to a quartz cuvette, aerobically, at the final concentration of 10 uM (protein), 50 uM (Fe(II)), and various Zn(II) concentrations (0, 10, 25, 30, 40, 50, 70 ,100 uM). Absorbance at 315 nm was monitored by UV-vis spectrophotometer every seconds for 1500 s by TimeDrive software. Negative controls were also included, assaying 50 uM Fe(II) in the absence of proteins to test background oxidation, and with each protein plus 100 uM Zn(II) to test the maximum concentration of zinc used i the assay.Data presented here represents three or more technical replicates per condition, means and standard deviations.Data from zinc inhibition experiments were also fitted using a Dose-Response (log(inhibitor) vs. response (three parameters) equation by GraphPad software to calculate IC50 and logIC50 values. Average of three or more technical replicates per condition was used as response.