AGE-RELATED-CHANGES IN HUMAN CERULOPLASMIN - EVIDENCE FOR OXIDATIVE MODIFICATIONS

Human plasma or serum from donors of age comprised between 15 and 95 y ears was analyzed for paramagnetic and total copper content, as well a s for immunoreactive ceruloplasmin content and oxidase activity. All p arameters were essentially unaltered, except the paramagnetic copper c ontent, which increased 2-fold upon aging. A dramatic change of the el ectron paramagnetic resonance spectrum due to ceruloplasmin occurred i n individuals over 65 years old and was associated with both an increa se of the type 1 copper signal intensity and the appearance of new res onances of a type 2 copper species. Ceruloplasmin was isolated from ei ther young or old donors. Spectroscopic analyses of the isolated prote ins confirmed the tendency of type 1 copper to stay reduced in the ''y oung'' and oxidized in the ''old'' protein. The type 2 copper signal o bserved in most young ceruloplasmin samples was different from the spe cies invariably present in the old protein. The magnetic parameters of the latter species were more consistent with a partially reduced trin uclear copper site. In vitro limited proteolysis resulted in identical fragmentation patterns and kinetics in both proteins. However, change s of the net electric charge were detected in the fragments of the pro tein isolated from aged individuals, which exhibited a carbonyl conten t of 0.6 mol of carbonyl/mol of protein. The same pattern of modificat ions, including a higher carbonyl content (0.65 versus 0.2 mol of carb onyl/mol of protein), could be reproduced by exposure of the young pro tein to the metal-catalyzed oxidation system iron/ascorbate. These res ults suggest that during aging ceruloplasmin is subjected to oxidative modifications which are likely to be the source of conformational cha nges around the copper sites leading to an intramolecular electron rea rrangement among the various copper sites.