Identification and Purification of a Non-Ceruloplasmin Ferroxidase of Human Serum

A non-ceruloplasmin ferroxidase (ferroxidase-II) protein was isolated from human serum and completely resolved from ceruloplasmin by DEAE-Sephadex A-50 chromatography. This protein was isolated and purified approximately 500-fold by utilization of the Cohn IV-1 fraction, DEAE-Sephadex A-50 chromatography, and gel filtration on Agarose A-15m, followed by gel filtration on Agarose A-50m. Ferroxidase-II differed from ceruloplasmin in many respects. Three of the most important differences were that ferroxidase-II (a) was yellow rather than blue as ceruloplasmin, (b) it was not inhibited by azide, and (c) it exhibited no p-phenylenediamine oxidase activity. Ferroxidase-II also differed significantly from the known plasma amine oxidases. The ferroxidase activity of ferroxidase-II was not lost by dialysis or ultrafiltration, but was inactivated by heat treatment and was proportional to the protein concentration at all stages of purification. Oxygen consumption with ferroxidase-II was observed simultaneously with iron oxidation. The ferroxidase-II activity in Wilson's disease serum represented a much larger percentage of the total ferroxidase activity than in normal serum. Although ferroxidase-II was decreased in Wilson's disease serum, it was reduced to a lesser extent than the ceruloplasmin ferroxidase activity. Thus ferroxidase-II may account for the lack of correlation of ferroxidase activity with p-phenylenediamine oxidase activity of Wilson's disease serum and may be responsible for the maintenance of near normal iron metabolism despite the low levels of ceruloplasmin.