A study of the kinetics of iron and copper binding to hen ovotransferrin

Abstract

The kinetics of iron and copper binding to hen's egg apoovotransferrin were studied by using citrate chelates of these metals at pH 9.3 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 a metal chelate to the protein and scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent color 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 coordination preferences, suggests that the process of metal binding or chromophore development for the 2 metal complexes must be similar.