The kinetics of iron and copper binding to hen9s-egg apo-ovotransferrin were studied by using citrate chelates of these metals at pH9.3 in borate buffer in the presence of bicarbonate. The kinetics of the absorbance change associated with the formation of the final product show a fast process, which is pseudo-first-order, where the reagents are in excess with respect to the protein, and the citrate concentration is higher than 25mM. At lower citrate concentration, the progress curves are clearly biphasic. There is marked dependence of the rate of the reaction on bicarbonate concentration, which may be interpreted as a displacement reaction of the ligand-metal-protein ternary complex. The kinetics have been interpreted in the framework of a reaction scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent colour development by displacement of the chelator by bicarbonate. The pH-dependence of this reaction supports the belief that tyrosine residues are involved in the process of iron-binding. The overall similarity of kinetics for iron and copper binding, notwithstanding their different co-ordination preferences, suggests that the process of metal-binding or chromophore development for the two metal complexes must be similar.
