Purification and characterization of the serum ferroxidase inhibitor.

The inhibitor of the serum ferroxidases, recently detected in rabbit serum, has been purified to homogeneity from human serum by a combination of gel-filtration and ion-exchange chromatography. The molecular weight, chromatographic behavior, electrophoretic mobility, electrofocusing pH, carbohydrate content, and reactivity with anti-human albumin during immunodiffusion indicate that the ferroxidase inhibitor is serum albumin. Copper-binding studies, proteolytic fragmentation studies, and a comparison of the inhibitory potencies of several albumin species which differ in their affinity for copper strongly indicate that albumin elicits its inhibitory effect on the serum ferroxidases by interacting with the functional copper of these enzymes. Kinetic analyses further suggest that albumin competes with substrate (ferrous iron) for binding to the functional copper of the serum ferroxidases.