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 cerulopl asmin (CP1; ferro-O-2-oxidoreductase, EC 1.163.1.) by dialyzing the en zyme 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 visibl e 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 t he sequence of CP depletion of certain copper ions. The same methods w ere applied in reconstitution studies to detect the return of differen t types of Cu2+. The experiments were performed on CP samples differin g 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 pr esence or absence of type 1 and type 2 copper ions. The results obtain ed 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 acco mplishment by the enzyme of its multiple functions. (C) 1997 Elsevier Science Inc.