Allosteric effects of sulfonate anions on the rates of iron release from serum transferrin

Abstract

Serum transferrin is the protein that transports ferric ion through the bloodstream and is thus a potential target for iron chelation therapy. However, the release of iron from transferrin to low-molecular-weight chelating agents is usually quite slow. Thus a better understanding of the mechanism for iron release is important to assist in the design of more effective agents for iron removal. This paper describes the effect of sulfonate anions on the rates of iron removal from C-terminal monoferric transferrin by acetohydroxamic acid, deferiprone, nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid at 25 °C in 0.1 M N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (Hepes) buffer at pH 7.4. These ligands remove iron via a combination of pathways that show saturation and first order dependence on the ligand concentration. The kinetic effects of the anions methanesulfonate, methylenedisulfonate, and ethylenedisulfonate were evaluated. All these anions increase the overall rates of iron release, presumably by binding to an allosteric anion binding site on the protein. The two disulfonates produce a larger acceleration in iron release than the monosulfonate. More detailed studies using methylenedisulfonate show that this anion accelerates the rate of iron release via the saturation pathway. The addition of methylenedisulfonate results in the appearance of a large saturation pathway for iron release by NTA, which otherwise removes iron by a simple first-order process. The sulfonate group was selected for these studies because it represents an anionic functional group that can be covalently linked to a therapeutic ligand to accelerate iron release in vivo. The current studies indicate that the binding of the sulfonates to the allosteric site on the protein is quite weak, so that one would not expect a significant acceleration in iron release at clinically relevant ligand concentrations.