Fig. 2

Ferroxidase activity of uuferritin. a The expression and purification of recombinant uuferritin protein. M: Molecular weight marker; Lane 1 and 2: The uuferritin protein induced by IPTG; Lane 3: The uuferritin protein after purification through a Ni²⁺ column. b The Fe²⁺ binding characteristics of uuferritin were investigated using IMAC. Lane 1: The columns were charged with Fe²⁺ and the bound protein was eluted with EDTA. Lane 2: The columns were charged with Fe²⁺ and the bound protein was eluted with EQ buffer. Lane 3: The column was not charged with metal and was eluted with EQ buffer. Lane 4: The column was not charged with metal and was eluted with EDTA. The samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. c The intrinsic fluorescence of 4 μM uuferritin protein was detected when 200 μM FeSO₄ was added. d Spectrophotometric kinetic curve of Fe²⁺ oxidation by uuferritin. Reactions were performed in 50 mM MOPS-NaOH, pH 7.4, at 37 °C. Reactions were started by the addition of 100 μM FeSO₄; the formation of Fe core was monitored by measuring absorbance at 305 nm in a 0.5 cm cuvette. Symbols for conditions are as follows: squares, 0.5 μM uuferritin and Fe²⁺; circles, 0.5 μM uuferritin only; triangles, Fe²⁺ only