(P11-060-23) Binding Chromium(III) To Form Mixed Cr(III),Fe(III) Serum Transferrins

Objectives: Transferrin, Tf, the protein that transports iron as Fe(III) from the blood to the tissues via endocytosis, is believed to also transport chromium(III), Cr(III). Under physiological conditions, Tf binds and releases Cr(III) rapidly from Cr(III)₂-Tf; however, the major form of Tf in the bloodstream is mono-ferric Tf. Given the low concentration of Cr(III) in the bloodstream, the form of Cr(III)-containing Tf that is transported is probably monochromic, monoferric-Tf (Cr(III),Fe(III)-Tf). Given that Tf has two specific metal-binding sites, one in both its C-terminal and the N-terminal lobe, two forms of Cr(III),Fe(III)-Tf can form. The binding of Cr(III) to mono-ferric Tf to both forms of Cr(III),Fe(III)-Tf has been examined in detail for the first time.

Methods: Monoferric-Tf with Fe(III) in the C-lobe was prepared by adding Fe(III) as Fe(nitrilotriacetate)₃] at pH 7.4; monoferric-Tf with Fe(III) in the N-lobe was generated by adding ferrous ammonium sulfate at pH 5.9. The pH of the solutions of monoferric-Tf’s in 0.1 mM Hepes buffer with 25 mM HCO₃^- were adjusted to pH 7.4 before the addition of one equivalent of Cr(III) as Cr(III) nitrate. Ultraviolet/visible data were collected every 30 s after addition for kinetics experiments. To estimate binding constants, varying amounts of Cr(III) were added to the monoferric Tf’s; ultraviolet/visible data were collected 30 min after Cr(III) addition to guarantee equilibrium was achieved.

Results: The addition of Cr(III) to monoferric Tf’s in 100 mM HEPES and 25 mM bicarbonate solution, pH 7.4 resulted in a rapid binding of Cr(III) to the open metal-binding site of the Tf’s. The rates of binding of Cr(III) to the monoferric Tf’s are between the rates of Cr(III) binding to the two sites of apoTf. Titrations of the monoferric Tfs with Cr(III) indicate the tight binding on one Cr(III) in each case.

Conclusions: Mono-ferric Tf’s bind one equivalent of Cr(III) rapidly and tightly to form mixed Cr(III),Fe(III)-Tf’s. Cr(III) is probably transported as mixed Cr(III),Fe(III)-Tf’s.

Funding Sources: The University of Alabama Bioinorganic Chemistry of Chromium Research Fund