Bicarbonate and the binding of iron to transferrin.

spectroscopy and by measurements of proton magnetic relaxation rates under conditions in which carbon dioxide (and therefore bicarbonate) is excluded. Even in the absence of bicarbonate, specific binding of Fe3+ to transferrin may occur. Nitrilotriacetate, ethylenediaminetetraacetate, and oxalate were found capable of replacing bicarbonate to form colored complexes with iron and transferrin. Since the nitrilotriacetate and oxalate complexes, in particular, have optical absorption spectra similar to those of the usual iron-transferrinbicarbonate complex, their recognition depends on correlating electron paramagnetic resonance and optical studies. The electron paramagnetic resonance spectrum of the irontransferrin-nitrilotriacetate complex consists of a line, 30 gauss in width, centered near the apparent g value, 4.25, whereas the oxalate complex shows a broad absorption, from 700 to 1400 gauss. In contrast, the bicarbonate complex has a spectrum centered near the apparent g vslue, 4.22, with a total width of 120 gauss. This spectrum is not influenced by changes in pH in the range, 6.1 to 10.5. Of a number of metal-complexing agents tested, only those with 2 or more carboxyl groups were found capable of replacing bicarbonate to form ternary complexes with transferrin and iron. Azide and thiocyanate were found ineffective, while a loo-fold excess of cyanide did not perturb the spectroscopic properties of iron-transferrin-bicarbonate. These findings suggest that bicarbonate is not simply coordinated to iron in iron-transferrin-bicarbonate. This view is corroborated by proton relaxation rate measurements, which show little change between iron-transferrin and iron-transferrin-bicarbonate. Cu2+ was also found to bind specifically to apotransferrin in the absence of bicarbonate, as evidenced by the electron paramagnetic resonance spectrum of the Cu2+-transferrin complex, which is quite diBerent from the spectrum of the Cu2+-transferrin-bicarbonate complex. Ternary complex formation among Fe3+, transferrin, and