Copper proteins and ferroxidases in human plasma and that of wild-type and ceruloplasmin knockout mice.
In the blood plasma of humans and rats, ceruloplasmin is the major copper-binding protein and ferroxidase, accounting for 70% of the copper present in the plasma, with the rest binding primarily to albumin and a macroglobulin. Systematic studies with fresh plasma were carried out to compare what occurs in the mouse. C57BL6 mice had half as much copper and pPD (p-phenylene diamine) oxidase activity as humans and rats, 20-40% as much ferroxidase activity as humans (determined using three different assays) and less inhibition by azide. Plasma from ceruloplasmin knockout mice had no pPD oxidase activity, but retained >50% ferroxidase activity (which was not as affected by azide). Modelling of mouse ceruloplasmin against the known X-ray structure of human ceruloplasmin indicated subtle but potentially significant changes in the pPD- and azide-binding sites. Purification and in-gel assays after native PAGE confirmed that mouse ceruloplasmin had ferroxidase activity but revealed an additional ferroxidase in ceruloplasmin knockout mouse plasma, which is also seen in size-exclusion chromatography. In the wild-type mouse, the 'ceruloplasmin' peak contained approximately 55% of the total copper, but ceruloplasmin knockout plasma exposed a major additional peak (180 kDa) which co-eluted with ferroxidase activity. Two other ferroxidases (700 and 2000 Da) were also detected in mouse and human plasma. Mammalian blood thus contains copper components and ferroxidases not reported previously.
Gray
Lawrence W
LW
Department of Chemistry and Biochemistry, California State University, Fullerton, CA 92834-6866, USA.
Kidane
Theodros Z
TZ
Nguyen
Anh
A
Akagi
Sheryl
S
Petrasek
Kristina
K
Chu
Yu-Ling
YL
Cabrera
Anthony
A
Kantardjieff
Katherine
K
Mason
Andrew Z
AZ
Linder
Maria C
MC
D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D002570:Ceruloplasmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017433:Protein Structure, Secondary; D051381:Rats
D001386:Azides; D002352:Carrier Proteins; C023548:copper-binding protein; D002570:Ceruloplasmin