Copper depletion/repletion of human ceruloplasmin is followed by the changes in its spectral features and functional properties.
Copper ions of different types were gradually eliminated from ceruloplasmin (CP1; ferro-O2-oxidoreductase, EC 1.16.3.1.) by dialyzing the enzyme against KCN. Protein was sampled 2, 4, 6, 22, and 28 h after the dialysis started. Atomic absorption allowed us to estimate the amount of copper atoms per CP molecule. Light absorption in the UV and visible regions along with fluorescence and EPR spectra were also registered. Oxidase and dismutase activities of the enzyme were measured at each step. The combination of the data thus obtained allowed us to trace the sequence of CP depletion of certain copper ions. The same methods were applied in reconstitution studies to detect the return of different types of Cu2+. The experiments were performed on CP samples differing in the amount of copper still bound after CN- treatment. It is shown that the oxidase activity is efficiently brought back to CP if, after the dialysis against cyanide, the catalytic center had preserved its type 3 Cu2+. Dismutase activity of CP did not depend greatly on the presence or absence of type 1 and type 2 copper ions. The results obtained allow a more precise evaluation of the role of different types Cu2+ in the assembly of the complex catalytic center of CP and in the accomplishment by the enzyme of its multiple functions.
Vassiliev
V B
VB
Institute for Experimental Medicine of RAMS, St. Petersburg, Russia.
Kachurin
A M
AM
Beltramini
M
M
Rocco
G P
GP
Salvato
B
B
Gaitskhoki
V S
VS
D002570:Ceruloplasmin / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D011190:Potassium Cyanide / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet
D003300:Copper; D002570:Ceruloplasmin; D011190:Potassium Cyanide