The Kinetics and Mechanism of Iron (III) Exchange between Chelates and Transferrin

Abstract

The reaction of iron(III)-nitrilotriacetate (NTA) with transferrin was studied with a stopped flow spectrophotometer. In the presence of bicarbonate ion the time course of the reaction is biphasic. The first phase is complete within a few tenths of a second, while the second phase requires some 10 s. Although the first phase does not follow second order kinetics, the initial velocity is dependent on both the transferrin and iron(III)-NTA concentrations. The fraction of the absorbance change of the total reaction contributed by the first phase is about 60%, and is relatively constant. The second phase follows first order kinetics. Addition of excess NTA or bicarbonate has little effect on either phase of the reaction. In the absence of bicarbonate the reaction time course becomes monophasic. The absorbance change of the reaction is greater in the absence of bicarbonate, and corresponds closely with static spectra obtained under anaerobic conditions. Based on the data presented, a reaction sequence is proposed. In the presence of bicarbonate, the first phase represents the reaction of iron(III)-NTA with a transferrin binding site to form an intermediate complex involving NTA, iron(III), and transferrin. In the second phase the breakdown of this complex frees the NTA and results in the formation of the specific iron(III)-transferrin-bicarbonate product. In the absence of bicarbonate a stable complex of NTA, iron(III), and transferrin is formed. Possible structures for the kinetic intermediate and the bicarbonate-free complex are discussed.