Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers.
Ceruloplasmin was isolated to purity from chicken plasma by a single-step chromatography on amino-ethyl-derivatized Sepharose. Molecular mass, as estimated by nonreducing sodium dodecyl sulfate-electrophoresis, was approximately 140 kDa, slightly higher than that found for ceruloplasmins from other sources. Specific activity as p-phenylenediamine oxidase was five times higher than that reported for mammalian ceruloplasmins. The copper content was estimated to be 5.01 +/- 0.35 atoms per protein molecule, 50% of which was EPR-detectable. The EPR spectrum was completely devoid of any signal typical of the type 2 copper as seen in the other blue multicopper oxidases and in ceruloplasmin from mammalian species. Anaerobic reduction of chicken ceruloplasmin resulted in the disappearance of the 330 nm optical band typical of type 3 copper, which was followed by the appearance of an EPR signal typical of type 2 copper. Subsequently, the type 1 copper and finally the newly formed type 2 copper were reduced. The original optical and EPR spectra were recovered within few minutes upon exposure of reduced ceruloplasmin to air. It is concluded that in oxidized chicken ceruloplasmin type 2 copper interacts with the diamagnetic pair responsible for the 330 nm absorption in such a way as to become EPR-undetectable and that the interaction is relieved by reduction of the pair. Whether this interaction is intrinsically weaker in other blue oxidases and ceruloplasmins studied or is lost with standard preparation procedures remains to be established.
Calabrese
L
L
Department of Biochemical Sciences, Università La Sapienza, Roma, Italy.
Carbonaro
M
M
Musci
G
G
D000818:Animals; D002570:Ceruloplasmin / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008970:Molecular Weight; D012756:Sheep; D013053:Spectrophotometry
D003300:Copper; D002570:Ceruloplasmin