[Spectral studies of the ceruloplasmin active site during its copper repletion and depletion]

Ceruloplasmin (CP; ferro-O2-oxidoreductase, EC 1.16.3.1) was gradually depleted of different type Cu2+ by dialysis against KCN. Protein samples were taken 2, 4, 6, 22 and 28 hrs after the beginning of dialysis. The number of copper atoms per CP molecule was estimated using atomic absorption spectrophotometry. Light absorption in UV and visible regions, fluorescence and EPR spectra were also recorded. The experimental results allowed to trace the sequence of release of certain Cu2+ ions from the CP molecule. The same methods used in the course of CP active center reconstitution made it possible to determine the dependence of its repletion with certain Cu2+ on the degree of the preceding catalytic center destruction. It was shown that efficient recurrence of the CP oxidase activity is achieved if after dialysis against KCN the enzyme retains type III Cu2+ in its active center. The data obtained allow to specify more precisely the role of different types of Cu2+ in the assembly of the complex catalytic center of CP and in exercising by the enzyme of its multiple functions.