title: Iodination significantly influences the binding of human transferrin to the transferrin receptor.
abstract: The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor's ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor-ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of Kd=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of Kd=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3-5-fold to Kd=0.66 nM and Kd=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.
authors.affiliation: Institut für Laboratoriumsmedizin und Pathobiochemie, Medizinische Fakultät Charité der Humboldt-Universität zu Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353, Berlin, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.forename: Reinhard
authors.initials: R
authors.lastname: Gessner
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007455:Iodine / Q000737:chemistry*; D007457:Iodine Radioisotopes; D007504:Iron Radioisotopes; D008024:Ligands; D008433:Mathematics; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D007504:Iron Radioisotopes; D008024:Ligands; D011990:Receptors, Transferrin; D014168:Transferrin; D007455:Iodine