CHARACTERSTICS OF RAT CERULOPLASMIN FROM THE SERUM OF ANIMALS,
WHICH RECEIVED SALTS OF SILVER WITH FOOD
A. N. Skvortsov,1, 2 E. Yu. Ilyechova,2 E. A. Zatulovskiy,2 A. N. Saveliev,2
N. V. Tsymbalenko,3 M. M. Shavlovsky,3 L. V. Puchkova 2, 3
1 Institute of Cytology RAS, 2 St. Petersburg State Polytechnical University,
and 3 Research Institute of Experimental Medicine, St. Petersburg;
1 e-mail: skvor@mail.cytspb.rssi.ru,
colbug@mail.ru
Abiogenic Ag(I) ions have electronic structure, similar to Cu(I) ions and can compete with Cu(I) for binding
sites of proteins which transport copper from extracellular media to sites of cuproenzyme formation in the
cell. Rodents receiving Ag-salts with food develop extracellular deficiency of copper associated with ceruloplasmin
(Cp, the major copper-transporting protein in blood serum of vertebrates). The present work focuses on
the studies of biochemical and physicochemical properties of Cp, obtained from blood serum of rats, which received
AgCl with food for 4 weeks (Ag-rats). Cp-fractions from blood serum of Ag-rats (Ag-Cp) were obtained
by ion-exchange chromatography with stepped gradient of NaCl. Each fraction was tested for oxidase and ferroxidase
activities by direct measurement of catalytic activity in the gel, and for specific activity in holo-Cp in oxidation
of chromogenic substrate. Molecular mass, electrophoretic mobility and ratio of apo- and holo-forms in
Ag-Cp fractions were evaluated by immunoblotting. Ag-Cp samples did not contain products of spontaneous
partial proteolytic degradation, characteristic of holo-Cp samples. Fractions of Ag-Cp and holo-Cp (from blood
serum of control rats) were compared by optical spectra, tertiary structure, susceptibility to thermal denaturation,
and by atomic Cu and Ag content. Ag-Cp contained 1—2 % Cp, which is similar by spectral and catalytic
properties with holo-Cp. [Ag] : [Cu] ration in Ag-Cp samples was about 4 : 1. As evidenced by circular dichroism
and differential scanning calorimetric studies, the major apo-fraction of Ag-Cp lacked tertiary structure of
native Cp and was significantly misfolded, which might explain its resistance to spontaneous partial proteolytic
degradation.
Key words: copper metabolism, ceruloplasmin, Cu(I)/Ag(I) transport, Ag-ceruloplasmin, ion-exchange
chromatography, circular dichroism, scanning calorimetry
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