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Investigation of cytochrome complex with carbon monoxide by spectrophotometry and two-dimensional correlation spectroscopy

DOI 10.18127/j20700997-201902-06


M.A. Proskurnin – Ph.D. (Chem.), Professor, Department of Analytical Chemistry, Faculty of Chemical, Lomonosov Moscow State University
M.E. Muratova – Student, Department of Chemistry, Lomonosov Moscow State University
A.V. Brusnichkin – Post-graduate Student, Faculty of Chemical, Lomonosov Moscow State University
P.A. Gorkin – Post-graduate Student, Vernadsky Institute of Geochemistry and Analytical Chemistry of the RAS (Moscow)
D.S. Volkov – Ph.D. (Chem.), Junior Research Scientist, Department of Analytical Chemistry, Faculty of Chemistry, Lomonosov Moscow State University. Moscow
E.V. Proskurnina – Ph.D. (Chem.), Associate Professor, Department of Medical Biophysics, Faculty of Fundamental Medicine, Lomonosov Moscow State University; Senior Research Scientist, Radiologic Diagnostics Department, Medical Research and Education Center of the Lomonosov Moscow State University
Yu.A. Vladimirov – Full Member of the RAS, Head of the Department of Medical Biophysics, Faculty of Fundamental Medicine, Lomonosov Moscow State University

The conditions for the reduction of cytochrome c were selected. It was found that the reduction reaction with ascorbate proceeds slowly and a certain time is necessary for the reaction to proceed. It depends on the concentration of ascorbate. The optimum ratio of the concentrations of cytochrome c and ascorbate was selected. The optimum ratio was equal to 1: 5.
The effect of sodium dodecyl sulfate (SDS) on cytochrome c associated with the oxidation of reduced cytochrome, was inves-tigated. This effect is not described in the literature, and the ratio of concentrations of cytochrome and SDS is not justified. It has been shown experimentally that when the ratio of cytochrome c and SDS concentration is more than 1:80, the reduced cytochrome c is oxidized. This allows us to select the optimum ratio of the concentrations of cytochrome c and SDS, and a ratio of 1:50 was selected.
While bubbling CO into a solution of cytochrome c, the denaturation of the latter was observed, which is not mentioned in previous papers on cytochrome c-CO complex. It is concluded that it occurs at the interface. In a CO-saturated water (without gas bubbling), the complex is not formed. Because of this, when selecting the CO bubbling rate, it is necessary to take into account two opposite factors: the degree of formation of the complex, which increases with the rate and the denaturation of the protein, which also increases.
Two-dimensional correlation spectroscopy (2DCOS) is used to assess whether the proposed conditions correspond to the production of the CO complex of ferrocytochrome c only and to what extent protein denaturation occurs in these conditions – a process that cannot be unambiguously estimated from the spectra. The difference between the conditions for obtaining the complex in the presence of SDS and tetraoleoylcardiolipin are shown.
It was shown that the complex of cytochrome c with CO is unstable, the kinetics of its decomposition was measured, and the kinetic equation was obtained by approximating the obtained curve.

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