Kinetic studies of iron deposition in horse spleen ferritin using O 2 as oxidant

Abstract

An optical flow cell provided a means to conveniently measure the rate of successive Fe 2+ oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe 2+ oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe 2+/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH) 3 formation and the presence (>95%) of Fe(OH) 3 deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5–50 Fe 2+/HoSF established that the reaction is overall first order in Fe 2+ concentration. However, when full progress curves were analyzed at a variety of Fe 2+/HoSF ratios, two first-order reactions ( k 1∼0.035 s −1 and k 2∼0.007 s −1) were found to contribute to the overall Fe 2+ oxidation reaction. The proportion of the fast reaction increased with increasing Fe 2+/HoSF ratio until at ∼400, it was the dominant reaction. For the Fe 2+/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe 2+ oxidation. Comparison of successive additions of 1.0 Fe 2+/HoSF showed that oxidation of the first 8–10 Fe 2+ produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H +/Fe 2+ ratio confirmed this difference in behavior by giving an H +/Fe 2+ ratio of ∼1.0 below and 2.0 for ratios >30 Fe 2+/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.