Dissociation and reconstitution of human ferroxidase II

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Abstract
The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and Cu components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. Treatment with sodium dodecyl sulfate [SDS] results in the dissociation of ferroxidase II into 2 nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol or Cu and has a MW of 3.8-3.9 .times. 105. In contrast, S-II contains bound phospholipid, cholesterol and Cu and has a MW of 2.2-2.4 .times. 105. The lipid composition of S-II is identical with the native enzyme. SDS-free S-I exhibits no ferroxidase activity. Immediately following removal of SDS, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the SDS to yield a fully active enzyme which chemically appears identical with native ferroxidase II. The reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4.degree. C for 6 wk without any loss in activity. S-II, the Cu and lipid-containing subunit, is probably the catalytic subunit; S-I is probably essential for the stabilization of the enzymic activity of S-II.

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