Electron paramagnetic resonance and difference ultraviolet studies of Mn 2+ binding to serum transferrin

Abstract

Serum transferrin is the mammalian protein whose normal function is to transport ferric ions through the blood among sites of absorption, storage, and utilization. It has two specific metal-binding sites that bind a variety of metal ions in addition to ferric ion. The macroscopic equilibrium constant for the binding of the first equivalent of Mn 2+ to apotransferrin has been determined by electron paramagnetic resonance spectroscopy (EPR) to be logK M1 = 4.06 ± 0.13 at pH 7.4 in 0.1 M N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (Hepes). An equilibrium constant for nonspecific binding of Mn 2+ to apotransferrin of logK ns = 2.93 ± 0.13 has also been obtained by using EPR. Binding of Mn 2+ to apotransferrin and to both C- and N-terminal nonferric transferrin has also been studied by difference UV spectroscopy. The second stepwise macroscopic equilibrium constant for the formation of Mn 2Tf is logK M2 = 2.96 ± 0.13. The site-specific microconstants for Mn 2+ binding are logK N = 3.13 ± 0.09 for the N-terminal site and logk C = 3.80 ± 0.09 for the C-terminal site. There does not appear to be any significant cooperativity between the two sites with respect to metal binding. An equilibrium model for the speciation of Mn 2+ in serum has been developed which estimates that almost 90% of Mn 2+ is bound to serum proteins, but only ∼ 1% is bound to transferrin. The weak binding of Mn 2+ to apotransferrin and the obvious inability of transferrin to compete with albumin indicates that the appearance of Mn-transferrin as a major serum species in vivo must involve oxidation of the metal to form the much more stable Mn 3+-transferrin complex. The computer model confirms that albumin has a sufficient binding affinity to complex most of the Mn(II) in serum in competition with the common low molecular weight ligands in serum. However, there is insufficient data to rule out the possibility that some other protein, such as α 2-macroglobulin, may compete with albumin for Mn(II).