Evaluation of iron chelating properties

The iron chelation capacity of the novel mitochondria-targeted antioxidants was evaluated by the spectrophotometric ferrozine method using a BioTek Synergy HT plate reader, by measuring the absorbance of the [Fe(Ferrozine)₃]²⁺ complex at 562 nm⁵⁰. The assay was performed in ammonium acetate buffer (pH 6.7) using a solution of ammonium iron (II) sulphate in ammonium acetate as the source of ferrous ions. In each well, a solution of the test compound (100 µM) plus ammonium iron (II) sulphate in ammonium acetate (20 µM) were added, incubated for 10 min and the absorbance was read at 562 nm. An aqueous 5 mM solution of ferrozine was freshly prepared and then added to each well (96 µM final concentration). After a new incubation at 37 °C for a 10 min period, the absorbance of [Fe(ferrozine)₃]²⁺ complex was measured at 562 nm. Blank wells were ran using DMSO instead of the test compounds. All compounds (protocatechuic and gallic acids, benzoic derivatives, EDTA and MitoQ₁₀) as well as ferrozine, were used at a final concentration of 100 µM. The absorbance of the first reading was subtracted to the final values to discard any absorbance due to the test compounds. Data are means ± SEM of three independent experiments and are expressed as % of Fe(II) chelation (EDTA = 100%). EDTA, used as reference, was found to chelate all available iron as it completely inhibited the formation of the coloured ferrozine-fe(II) complex.