The nonspecific binding of Fe3+ to transferrin in the absence of synergistic anions.

Gw Bates,Mr Schlabach

doi:10.1016/s0021-9258(19)41698-0

Abstract

An obligatory role for barbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. There are an equal number of confirmatory and negative reports of specific Fe3+-transferrin binary complexes. A criticism of previous studies is the use of only one synthetic route, and limited product testing. This study reports the development of several preparative routes aimed at the formation of a specific Fe3+-transferrin complex, and the characterization of the products by spectrophotometry and chemical reactivity. The preparative routes described include: (a) displacement of carbonate from Fe3+-transferrin-CO32- at low pH followed by removal of CO2 by several techniques; (b) addition of FeCl3 to apotransferrin under CO2-free conditions; (c) oxidation of Fe2+ in the presence of apotransferrin under CO2-free conditions; (d) reaction of apotransferrin with nonsubstituting Fe3+ complexes in the absence of CO2; and (e) attempts to displace anions from weak Fe3+-transferrin-anion complexes. The product were examined with regard to their visible spectra, and their examined with regard to their visible spectra, and their reactivity with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) citrate. The results are compared with the characteristics of Fe3+-transferrin-anion complexes and nonspecific Fe3+, transferrin mixtures. The data indicate that in the absence of synergistic anions the affinity of the specific metal binding sites of transfe-rin for Fe3+ is so low as to not compete favorably with hydrolytic polymerization and nonspecific binding effects.

Highlights

In this publication we report the use of several synthetic routes aimed at the formation of a binary Fe3f-transferrin complex, and the development of techniques to test the nature of the products
The results presented below indicate that carbonate or other synergistic anion is required for FeJf binding at two specific metal binding sites of the transferrin molecule
It is apparent that the reaction of the Fez+, transferrin mixture with NaHC03 is the same as that characteristic of the reaction of vacant sites with nonspecifically bound iron, and quite different from the reactivity of weak Fez+-transferrin-anion complexes

Summary

SUMMARY

An obligatory role for carbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. Fraenkel-Conrat (8) presented spectrophotometric evidence for a COS-free iron-conalbumin complex This was later repudiated by Warner and Weber (9) who suggested that there is no iron binding in the absence of CO% Aisen et al (2) attempted to solve the conflict by preparing a mixture purported to be C02-free transferrin with bound iron. Ferric salts give a poor yield of complexed iron even in the presence of CO2 (12) Another criticism regards the heavy reliance on “color formation” or the appearance of an EPR signal as an index of success. In this publication we report the use of several synthetic routes aimed at the formation of a binary Fe3f-transferrin complex, and the development of techniques to test the nature of the products.

PROCEDURES

RESULTS

DISCUSSION