| Abstract
| - The exchange of Fe3+, Tb3+, In3+, Ga3+, and Al3+ between the C-terminal metal-binding site of the serum irontransport protein transferrin and the low-molecular-mass serum chelating agent citrate has been studied at pH 7.4and 25 °C. The removal of Ga3+, In3+, and Al3+ follows simple saturation kinetics with respect to the citrateconcentration. In contrast, removal of both Fe3+ and Tb3+ shows a combination of saturation and first-order kineticbehavior with respect to the citrate concentration. The saturation component is consistent with a mechanism formetal release in which access to the bound metal is controlled by a rate-limiting conformational change in theprotein. The first-order kinetic pathway is very rapid for Tb3+, and this is attributed to a direct attack of the citrateon the Tb3+ ion within the closed protein conformation. It is suggested that this pathway is more readily availablefor Tb3+ because of the larger coordination number for this cation and the presence of an aquated coordination sitein the Tb3+−CO3−Tf ternary complex. There is relatively little variation in the kmax values for the saturation pathwayfor Tb3+, Ga3+, Al3+, and In3+, but the kmax value for Fe3+ is significantly smaller. It is suggested that protein interactionsacross the interdomain cleft of transferrin largely control the release of the first group of metal ions, while thebreaking of stronger metal−protein bonds slows the rate of iron release. The rates of metal binding to apotransferrinare clearly controlled in large part by the hydrolytic tendencies of the free metal ions. For the more amphotericmetal ions Al3+ and Ga3+, there is rapid protein binding, and the addition of citrate actually retards this reaction. Incontrast, the nonamphoteric In3+ ion binds very slowly in the absence of citrate, presumably due to the rapidformation of polymeric In−hydroxo complexes upon addition of the unchelated metal ion to the pH 7.4 proteinsolution. The addition of citrate to the reaction accelerates the binding of In3+ to apoTf, presumably by formingsoluble, mononuclear In−citrate complexes.
- The rates of exchange of Fe3+, Tb3+, In3+, Ga3+, and Al3+ between the C-terminal metal-binding site of serum transferrin and citrate have been measured at pH 7.4 and 25 °C. The predominant pathway for metal ion release exhibits saturation kinetics with respect to the citrate concentration. The rate of this process is relatively constant for all the metal ions except Fe3+, which is released much more slowly. The rate of binding of these metal ions to apotransferrin is strongly affected by their hydrolytic behavior. The amphoteric metal ions Ga3+ and Al3+ bind to apotransferrin relatively rapidly. In contrast, protein binding of the nonamphoteric In3+ ion is extremely slow in the absence of citrate.
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