Dissociation and reconstitution of human ferroxidase II.
The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper 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. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, 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 sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity.
D001051:Apoenzymes; D003300:Copper / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D008055:Lipids / Q000032:analysis; D046911:Macromolecular Substances; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism; D011489:Protein Denaturation; D014168:Transferrin
D001051:Apoenzymes; D008055:Lipids; D046911:Macromolecular Substances; D014168:Transferrin; D003300:Copper; D007501:Iron; D010088:Oxidoreductases; C023100:ferroxidase II