EPR signal, purple color, and iron binding in porcine uteroferrin.

Titration of purple uteroferrin with two reducing equivalents of ferrous ion doubles the intensity of its g' = 1.74 EPR signal while inducing only minor changes in spectral characteristics. Unexpectedly, intensification of the EPR signal is not accompanied by a commensurate increase in visible absorption: only a small shift in peak position from 545 to 525 nm is observed. These observations suggest that uteroferrin can bind a second iron to form a paramagnetic complex that is essentially nonchromophoric. Titration of the pink protein with one oxidizing equivalent of ferric ion also doubles the intensity of its g' = 1.74 EPR signal, again shifting the primary visible absorption band to 525 nm. In either case, therefore, it is possible to augment the g' = 1.74 EPR signal without a corresponding augmentation of purple-pink color. Conversely, the addition of hydrogen peroxide (but not ferricyanide) to purple uteroferrin obliterates the g' = 1.74 EPR signal without abolishing the protein's visible absorption spectrum. Hence, it is also possible to have purple color without a corresponding g' = 1.74 EPR signal. To explain these curious results, two possible models, one involving low spin ferric iron exclusively and the other a combination of low spin ferric and high spin ferrous, are adduced.