Determination of the Binding Constant of Terbium-Transferrin

Apotransferrin (apo Tf) in 0.1 M N-(2hydroxyethyl)piperazine-N2-ethanesulfanic acid at 25 ̊C and pH 7.4 has been titrated with acidic solution of Tb. The binding of Tb at the two specific metal-binding sites of transferrin was followed from the changes in the difference UV spectra at 245 nm. The molar absorptivity per binding site for Tb-Tf is 22,500 ± 1000 Mcm. To determine the Tb-Tf binding constants, apo Tf was titrated with Tb solutions which also contained nitrilotriacetic acid as a competitive chelating agent. The sequential macroscopic equilibrium constants for the binding of two metal ions are log K1 = 9.96 ± 0.38 and log K2 = 6.37± 0.38. Titrations of both C-terminal and N-terminal monoferric transferrin with Tb indicate that terbium binding is stronger at the C-terminal binding site. The value of K1 for Tb is substantially higher than the teransferrin binding constants reported for larger lanthanides. It is possible that there are steric interferences to the binding of larger lanthanides. However, an analysis of the transferrin binding constants using linear free energy relationships for metal complexation suggests that the metal ionic radius alone is not the major determining factor. A change in the number of coordinated water molecules in the aqueous ions for Tb compared to the larger lanthanides may be a more important factor.