Stoichiometry of iron binding by uteroferrin and its relationship to phosphate content.
The analysis of uteroferrin's iron content by an acid-release method used in previous studies shows a critical dependence on phosphate, i.e. the native phosphate-free enzyme yields two irons/molecule, while enzyme with one tightly bound phosphate gives closer to one iron. In contrast, two irons/molecule of protein are found in samples assayed by a wet ash method. When iron assays are carried out on samples of purple two-iron protein reductively stripped of their phosphate, both methods again yield two iron atoms/molecule. However, the discrepancy between the two methods recurs when phosphate is added to samples of pink protein which were formerly free of phosphate. These results suggest that phosphate bound to native uteroferrin may have interfered with iron determinations in some earlier studies. Furthermore, enzyme samples with one tightly bound phosphate have the optical purity index (i.e.A280/A545 approximately less than 14.0) and extinction coefficient at 280 nm, characteristic of putative one-iron preparations. There is little doubt, therefore, that previous EPR, magnetic susceptibility, and iron titration experiments thought to have been carried out on genuine one-iron preparations were in fact done on samples of two-iron protein bearing a single tightly bound phosphate. Reassessment of earlier studies indicates that the properties of putative one-iron preparations may be reconciled with those of the two-iron phosphate-laden protein studied here.
D000135:Acid Phosphatase; D000818:Animals; D005260:Female; D007501:Iron / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase
D007527:Isoenzymes; D008667:Metalloproteins; D010710:Phosphates; D007501:Iron; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase