Speciation 98: Abstracts

Transferrin is the iron transport protein in human serum. It consists of two similar lobes, each of which contains a single high-affinity metal binding site. Since the protein is not normally saturated with ferric ion, it is a potential transport protein for other metal ions that might enter the blood. Transferrin is capable of binding a very wide variety of di-, tri-, and tetravalent metal ions in vitro. However, the binding affinities for divalent ions are generally rather small, so that in vivo binding is probably restricted to tri- and tetravalent metal ions. Transferrin is the primary serum chelating agent for Fe³⁺, Ga³⁺, Al³⁺, In³⁺, Mn³⁺, and Pu⁴⁺.

The unique feature of the transferrins is the critical role of a synergistic carbonate anion in the metal-binding process. Metal complexation actually proceeds in two steps.

apoTf + CO32- CO32--Tf	(1)
M3+ + CO32- M3+-CO32--Tf	(1)

As a result of these sequential equilibria, the effective metal-transferrin binding constants are dependent upon the solution carbonate concentration. This can have a significant effect on in vitro studies, in which the carbonate concentration can vary widely. Under physiological conditions the serum carbonate concentration is high enough to push reaction (1) almost to completion, so that transferrin operates in vivo near its maximum binding affinity.

Binding constants for Fe³⁺ and the trivalent group 13 metal ions fall in the order Fe³⁺ > Ga³⁺ > In³⁺ >> Al³⁺. This is the usual trend observed for these metal ions with low molecular weight chelating agents. The rate of binding of the aquo metal ions to transferrin is quite rapid. However, large variations in the apparent rate of binding are observed due to variations in the hydrolytic tendencies of these metal ions. At neutral pH both Fe³⁺ and In³⁺ hydrolyze very rapidly to polymeric hydroxide complexes, and subsequent transfer of the metal ion to transferrin can be very slow. These metals must be delivered to the protein by a modest chelating agent such as nitrilotriacetic acid or citrate. Both Ga³⁺ and Al³⁺ are amphoteric and form soluble, monomeric M(OH)^4- anions at physiological pH. These anions readily react with the protein, so that Ga and Al can be easily added to transferrin as simple inorganic salts. Competition from hydrolysis affects binding affinities as well. The order of the "effective" binding constants for pH 7.4 is Fe³⁺ > In³⁺ > Al³⁺ > Ga³⁺. Both the C- and N-terminal binding sites can be saturated with Fe³⁺ and In³⁺, but not with Al³⁺ or Ga³⁺.

The in vivo binding of Mn follows a different pattern. Manganese is usually injected as Mn²⁺, which binds very weakly to transferrin and albumin. The labile Mn²⁺ ion is rapidly cleared from the blood by the liver. A small fraction of the manganese is oxidized to Mn³⁺, bound very tightly to Tf, and released back into the blood for delivery of Mn to extra-hepatic tissues.

The rates of metal release from transferrin are controlled in large part by a conformational change in the protein. The rates vary strongly with the strength of the metal-transferrin binding, which suggests that the conformational change may involve breaking one or more metal ligand bonds. In vivo metal release is associated with binding of the metal-transferrin complex to the transferrin receptor. Receptor mediated cellular uptake is important for Fe³⁺, Ga³⁺, Al³⁺, and Mn³⁺, but not for In³⁺.

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