Physical and chemical studies on ceruloplasmin. IV. Preparation of radioactive, sialic acid-free ceruloplasmin labeled with tritium on terminal D-galactose residues.

Ceruloplasmin, a normally occurring plasma protein containing copper and carbohydrate as integral parts of its molecule, appears to play some role in maintaining the normal, near zero balance of body copper (1). However, despite considerable experimental effort, little definitive information is currently available concerning either this role or the metabolism of the protein. The present investigation was undertaken in an attempt to provide a derivative of ceruloplasmin, labeled with a long lived isotope, which would be capable of serving as a reagent in subsequent experiments in vivo directed toward these two questions. Recent studies by Jamieson (2) have indicated that each molecule of ceruloplasmin possesses 9 to 10 carbohydrate chains consisting of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine. In addition, Jamieson isolated several glycopeptide fractions of ceruloplasmin in which the sialic acid was shown to be terminal, since it could be quantitatively removed by neuraminidase. By analogy with a number of other plasma glycoproteins in which it has been demonstrated that sialic acid is bound glycosidically to D-galactose (3), it seemed possible that the galactose, exposed by the action of neuraminidase upon ceruloplasmin, could then be oxidized at unsubstituted carbon 6 positions, by the highly specific enzyme, D-galactose oxidase (4-6). Reduction, with tritiated borohydride, of the aldehyde thus