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Highly purified human ceruloplasmin was isolated from fresh donor blood in the presence of inhibitors of proteolysis and from stored retroplacental blood serum both with and without inhibitors of proteolysis. According to the data of electrophoresis. ultracentrifuge sedimentation velocity and sedimentation equilibrium, all the ceruloplasmin samples were homogeneous, their molecular weight being 130 000. The dissociation of the samples treated by dodecylsulphate, guanidine · HCl, and urea was studied by means of quantitative analytica and preparative electrophoresis, and sedimentation equilibrium. The dissociation patterns depended on whether inhibitors were used in the isolation procedure. Polypeptides with molecular weights of 130 000, 112 000 and 16 000 (minor component) were obtained, if phenylmethylsulfonyl fluoride and/or 6‐aminohexanoic acid were used; if these compounds were not used, the dissociation yielded additional polypeptides of molecular weights of 100 000 (minor component), 64 000 and 48 000. Under proteolysis‐favouring conditions the relative amount of these polypeptides increased. Prolonged storage of samples under sterile conditions without inhibitors of proteolysis resulted in a decrease of the relative amount of polypeptides with molecular weights of 130 000, 112 000, 100 000, 64 000 and 48 000, contrary to that of the 16 000‐Mr polypeptide, which increased. At the same time new polypeptides appeared with molecular weights of 42 000 and 21 500–23 000. Spontaneous specific fragmentation of the ceruloplasmin molecule is due to trace amounts of proteases, which seem to originate from blood plasma. Limited tryptic hydrolysis of the ceruloplasmin globule resulted in the appearance of polypeptides with the same molecular weights which were observed in spontaneous fragmentation. A conclusion is drawn that the ceruloplasmin molecule in vivo is a single polypeptide chain with at least five bonds which in vitro are the points of specific proteolytic fragmentation, yielding six principal fragments.

The investigation of human ceruloplasmin by spectral methods (EPR and spectrophotometry) demonstrated that type 2 Cu2+‐containing centres occur not in one, but in two stable forms, differing in EPR and optical spectra. The differential optical spectra of these forms were recorded and the differences in molar absorption coefficients determined. By the EPR method, it was shown that both forms of these centres exist in the blood serum of control donors, as well as in the serum of patients. The relative content of these forms depends on the organism physiological state or on the presence of some pathological condition. The ferroxidase activity of ceruloplasmin against hemoglobin was proved spectrophotometrically. The involvement of other serum proteins in this process cannot be ruled out. The conformational state of ceruloplasmin molecules plays an essential role in its oxidase activity.

Comparative immunochemical analysis of ceruloplasmin-synthesizing polyribosomes in liver biopsies from control subjects and homozygous carriers of the Wilson's mutation was performed. According to I125-antibody binding data, the amount of ceruloplasmin-forming liver polysomes in patients with Wilson's disease was 10--20 times lower than that in non-Wilson patients. Correspondingly, the pulse labeling of ceruloplasmin polypeptides was decreased several-fold in the cell-free liver preparations from patients with Wilson's disease.

The review presents both our own and literature data on studies of pathways of evolution of the so-called multinuclear blue copper-proteins (MBCP) that have the domain organization. The MBCP are widely spread in living nature, they have been revealed in cells of archei, bacteria, and eukaryotes. The MBCP composition includes the copper-proteins such different by their properties as oxidases, reductase, blood coagulation factors V and VIII. Most likely, MBCP have been originated from a low-molecular protein-precursor similar topologically with the blue electron-transporting protein of the cupredoxin type, as a result of action of various evolutionary mechanisms: amplification of genes, formation of protein structures by different combinations of domains, a change of size of domains, the segment elongation at the expense of the activational domain, formation and loss of copper-binding centers, variation of amino acid ligands in such centers, the appearance of centers of binding of other proteins, glycosylation, etc.