Ferrous Ion Oxidation Assay Protocol

The assay is conducted at 30 °C by adding Fe²⁺ to a mixture of protein/enzyme (0.8 uM), Tris-HCl buffer (50 mM, pH 7.0), and (NH₄)₂Fe(SO₄)₂ (40 µM). A 10-µl aliquot is taken at 5 min after the assay reaction started and mixed with a 100-µL solution of Xylenol Orange (XO) (125 µM) and H₂SO₄ (25 mM). After incubation at room temperature for 5 min, the concentration of ferric ions is determined by measuring the absorbance at 595 nm using a TECAN microplate reader (Infinite M200 Pro, TECAN, Zürich, Switzerland) with i-control v1.7 software. The auto-oxidation of the ferrous ions is measured under the same assay conditions but without adding protein/enzyme.