Equal 140 μL volumes of weakly acidic
FeSO4 solution containing 96 iron/protein shell and buffered apoferritin
solution were mixed at 25 °C in the thermostated sample compartment
containing a 280 μL quartz stopped-flow cuvette with 1 cm optical path
length. Iron oxidation experiments were performed by measuring the
absorbance at 300 nm as previously reported (Deng, Liao et al., 2010).
Datawere acquired every 12.5ms (the shortest acquisition time possible
of the Cary 50).
Iron reductive release was investigated using ferrozine as the Fe2+
chelating as previously reported (Deng et al., 2010). Briefly, the development
of [Fe(ferrozine)3]2+ wasmeasured by recording the increase in absorbance
at 562 nm using a Varian Cary 50 spectrophotometer (Varian,
USA). The rate of iron release from holoferritin (800 Fe3+/protein shell)
was estimated using ε562 = 27.9mM−1 cm−1 as previously described
(Deng, Cheng et al., 2010), and the kinetic datawere analyzedwith Origin
8.0 software (Microcal Inc.).