Kinetics of iron removal from monoferric and cobalt-labelled monoferric transferrins by ethylenediaminetetra(methylenephosphonic acid) and ethylenediaminetetraacetic acid

Abstract

The rates at which ferric ion is removed from the two metal binding sites of the serum iron transport protein transferrin by ethylenediaminetetra(methylenephosphonic acid) (EDTP) and ethylenediaminetetraacetic acid (EDTA) have been evaluated. Solutions were buffered at pH 7.4 by 0.1 M N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid and maintained at 25°C. Pseudo first-order rate constants as a function of ligand concentration were measured for iron removal from diferric transferrin, from N- and C-terminal monoferric transferrin and from monoferric transferrins which had been labelled at the vacant site with kinetically inert cobalt(III). The data on iron removal from the cobalt-labelled monoferric transferrins provide accurate estimates of the rate constants for iron removal from diferric transferrin. A significant negative cooperativity is observed, in which occupancy of the C-terminal site slows the rate of iron release from the N-terminal site. Iron release from most of the transferrin complexes shows a combination of saturation and first-order kinetics which are interpreted in terms of parallel pathways for iron removal. The addition of 100 mM perchlorate to the samples accelerates iron removal through both pathways for the C-terminal site, but sharply decreases the rate of iron removal from the N-terminal site. The implications of these new results with respect to proposed mechanisms of iron release from transferrin are discussed.