Abstract
Human plasma or serum from donors of age comprised between 15 and 95 years was analyzed for paramagnetic and total copper content, as well as for immunoreactive ceruloplasmin content and oxidase activity. All parameters were essentially unaltered, except the paramagnetic copper content, which increased 2-fold upon aging. A dramatic change of the electron paramagnetic resonance spectrum due to ceruloplasmin occurred in individuals over 65 years old and was associated with both an increase of the type 1 copper signal intensity and the appearance of new resonances of a type 2 copper species. Ceruloplasmin was isolated from either young or old donors. Spectroscopic analyses of the isolated proteins confirmed the tendency of type 1 copper to stay reduced in the "young" and oxidized in the "old" protein. The type 2 copper signal observed in most young ceruloplasmin samples was different from the species invariably present in the old protein. The magnetic parameters of the latter species were more consistent with a partially reduced trinuclear copper site. In vitro limited proteolysis resulted in identical fragmentation patterns and kinetics in both proteins. However, changes of the net electric charge were detected in the fragments of the protein isolated from aged individuals, which exhibited a carbonyl content of 0.6 mol of carbonyl/mol of protein. The same pattern of modifications, including a higher carbonyl content (0.65 versus 0.2 mol of carbonyl/mol of protein), could be reproduced by exposure of the young protein to the metal-catalyzed oxidation system iron/ascorbate. These results suggest that during aging ceruloplasmin is subjected to oxidative modifications which are likely to be the source of conformational changes around the copper sites leading to an intramolecular electron rearrangement among the various copper sites.
Highlights
Human plasma or serum from donors of age com- plasma
In vitro limited proteolysis resulteidn identical frag-this protein (Malmstromet al., 1975),including the type mentation patterns and kinetics in both protHeionws.ever, changes of the net electric charge were detected in the fragments of the protein isolatedfromaged individuals, which exhibiteda carbonyl contentof 0.6 mol of carbonyl/mol of protein
The EPRsignal of the type 2 center would be an intrinsic feature of human CP, both in serum be reproduced by exposureof the young protein to the and in the purified protein (Rylkov et al, 1991).In a series of metal-catalyzoedxidatiosynsteimron/ascorbate
Summary
Disclosure, to a various extent, of type 2 copper signals in the EPR spectrum This has been shown to occur upon storage of the protein or during redox cyclesinvolving electron transport from donor substrates to oxygen (Calabrese et al, 1988, Ceruloplasmin (CP),’ theblue oxidase present in all verte- 1989). In this latter case, the type 2 signal behaves brates, is the major copper-containing protein of human as a transient and possesses slightly different magnetic pa-. Isoelectric focusing in the presence of 9 M urea was performed in a Pharmacia Multiphor I1 apparatus on ultrathin
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