Kinetics of iron removal from human serum monoferric transferrins by citrate

Abstract

The kinetics of release of Fe3+ from human serum diferric transferrin (Fe2Tf) and the Nand C-terminal monoferric transferrins (FeNTf and FkTf) to citrate were studied spectrophotometrically a t pH 7.4,37 OC, and 1.30 M ionic strength. Due to the similarity in rate constants for iron release from the two sites, iron release from Fe2Tf appears to be a monophasic process. Under the present experimental conditions, the C-terminal site is somewhat more labile than the N-terminal site. The observed rate constants for iron release from FeNTf and FecTf show a dependence on citrate concentration (at constant ionic strength) which changes from one apparent linear gradient at low citrate concentration to a second, smaller, apparently linear gradient a t higher citrate concentration. This is described by an equation of the form kob = (k,[Cit][X] + k2K[CitI2]/([X] + K[Cit]}, where k, and k2 are the second-order rate constants for pathways that are dominant a t low and high citrate concentration, respectively, and K is an equilibrium constant reflecting the competition by citrate and an anion, X, from solution for anion-binding sites, distinct from the synergistic-anion-binding sites, which control the kinetics of iron release. The experimental data both for FecTf and FeNTf are adequately fitted by assuming the existence of one such site. The effect of the anions HPO:-, CIOL, Cl-, and SO,*on the kinetics was studied, and it was shown that phosphate, while itself only mobilizing iron very slowly from transferrin, has a marked accelerating effect on metal release to citrate. The present results are compared to the previously reported results for release of AI3+ from AI2Tf to citrate.