Spectroscopic evidence for a difference between the iron-binding sites of conalbumin.

Conalbumin complexes containing one, and only one, specifically bound metal ion were isolated by the isoelectric focusing technique. This made possible the preparation of a series of mixed metal or mixed isotope complexes: GaoFeiconalbumin, Fe,Gaiconalbumin, S6Fezconalbumin, 5Te05Teiconalbumin, “FeoS6Feiconalbumin, and 57Fezconalbumin. The appropriate species were studied by EPR, Mijssbauer, and optical spectroscopy. It was possible, in this way, to determine the contribution of each site to the spectrum of the protein. Occupancy of the first (or “inner”) binding site by Fe(III) produces EPR, MSssbauer, and optical difference spectra which are readily distinguishable from those given by the second (“outer”) site. Summation spectra obtained from the two sites are the same as those obtained from diferric conalbumin. We conclude, therefore, that the specific binding sites of conalbumin are not identical, and that the protein does not bind metal ions in a simple random fashion.