Studies on the oxidase properties of ceruloplasmin: factors in normal and Wilson's-disease serum affecting oxidase activity.

The oxidase activity of normal serum is due to the presence of the blue copper protein, ceruloplasmin. This is an a-globulin of molecular weight 151,000 containing 8 copper atoms per molecule (1). Its physiological substrate, if any, is unknown (2), but it has a weak oxidase activity for compounds that can be oxidized to quinone or quinone imines (3). The copper atoms in the protein appear to be arranged in functional pairs, and it seems probable that a specific function exists that will be shown to depend upon the unique chemical nature of the protein, as has already been demonstrated for another copper protein, tyrosinase (4). The concentration of ceruloplasmin in serum can be measured by estimating light absorption at 605 mp, before and after the destruction of the copper-protein bond with cyanide, by immunochemical techniques, or by measuring the oxidation of paraphenylene diamine (or related compounds) either in a Warburg flask or a spectrophotometer (4). These three methods all give comparable results, although measuring different properties of the protein: the copper-protein bond, the antigenic site, and the catalytic center. The most commonly used laboratory method to determine ceruloplasmin is the estimation of the color formed during the oxidation of paraphenylene diamine, or its dimethyl derivative (5-7). The result can be placed on a semiquantitative basis by comparison with those obtained by use of known amounts of purified ceruloplasmin, which within limits, gives an activity proportional to the amount of ceruloplasmin present (3). It has been suggested, however, that there are highand lowmolecular-weight compounds present in normal plasma that inhibit the oxidase properties of ceruloplasmin; Akerfeldt (8) believed the predominant one was ascorbic acid. Compounds with sulfhydryl (SH) groups have also been implicated (9) and so has serum albumin (10). Richterich (11) states that ceruloplasmin oxidase activity is inhibited 10 to 25% by serum. If this is so, it becomes necessary to add ceruloplasmin to serum to obtain accurate quantitative results. Scheinberg (12) has stated that purified ceruloplasmin added to Wilson's-disease serum, with essentially zero ceruloplasmin content, has its oxidase activity potentiated 25%o. This discrepancy suggests that inhibitors present in normal serum may be absent in Wilson's disease, or there may be factors present in Wilson's-disease serum able to potentiate the oxidase activity of normal ceruloplasmin.