The synergistic binding of anions and Fe3+ by transferrin. Implications for the interlocking sites hypothesis

The finding that transferrin does not bind Fe3+ at the specific metal binding sites in the absence of carbonate and synergistic anions emphasizes the fundamental importance of the anion binding site to the chemistry of Fe3+-transferrin-CO32-. An important question regards the chemical and structural requirements for carbonate substitution. This has been, however, an area of some dispute in the literature. We have utilized four synthetic routes for the preparation of Fe3+-transferrin-anion complexes. The products have been examined with regard to spectral properties, and reaction with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) sodium citrate under CO2-free conditions. The results provide information as to which anions are synergistic, and the basic properties of the Fe3+-transferrin-anion complexes that are formed. The 6 inorganic anions that were tested were all found to be nonsynergistic. Dihydroxyacetone and glyceraldehyde were also nonsynergistic. Dicarboxylic acids were found to form stable Fe3+-transferrin-anion complexes which were only slowly displaced by carbonate. Several monocarboxylic acids with proximal aldehyde, ketone, alcohol, amino, or thiol functional groups proved to be synergistic. CPK molecular model studies suggested the functional group and the carboxylic acid must be able to fit within a site between 6.3 and 7.0 A in maximal length. One large substituent could be accommodated by the site, however, two methylgroups on the alpha carbon to a carboxylate group could not be accommodated. Chloroacetate and monocarboxylic acids were nonsynergistic. The results are interpreted in terms of an interlocking sites hypothesis which envisions the synergistic anion as interacting with the protein via its its carboxyl group and bonding with the Fe3+ via its proximal functional group.