Stoichiometric or thermodynamic equilibrium constants for the binding of Fe3+ to transferrin were evaluated by the method of equilibrium dialysis using citrate as a competing complexing agent, near pH 6.7 and near pH 7.4. In each case K1 was substantially greater than K2, the effect being more marked at lower pH. These overall stability constants for the binding of iron, which take account of the participation of bicarbonate and protons in the reactions of binding, decrease with increasing pH. This may reflect the influence of the protein's negative charge, which increases with pH, on the critical role of the anion in metal binding. However, at any pH and pCO2 apparent stability constants may be defined, and these increase with increasing pH as a result of the inverse dependence of iron binding on the fourth power of the hydrogen ion concentration. At pH 7.4 and atmospheric pCO2, the apparent stability constants K'1 and K'2 are 4.7 x 1020 M-1 and 2.4 x 1019 M-1, respectively. Using the urea gel electrophoresis method of Makey and Seal (Makey, D.G., and Seal, U.S. (1976) Biochim. Biophys. Acta 453, 250-256), relative concentrations of the two species of monoferric transferrin were measured in each preparation at equilibrium. This made it possible to estimate the four intrinsic site constants for the binding of iron to transferrin. A slight negative cooperativity was evident in the binding of iron to each site. Although one site, designated the a-site, is more strongly binding than the b-site, it is not necessarily more accessible to all complexes of iron. Thus, iron as ferric citrate, ferric oxalate, ferrous ammonium sulfate and ferric chloride preferentially occupy the b-site when presented to transferrin under conditions in which an equilibrium distribution of iron between the binding sites of the protein would not be expected. Iron as ferric nitrilotriacetate, however, is directed toward the a-site. This is also the site which retains iron at low pH. On the basis of these observations single site transferrins were prepared, and a difference in their EPR spectra was demonstrated under near physiologic conditions.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1978|
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