Biomedical Chemistry: Research and Methods 2024, 7(3), e00225

New Highly Sensitive Methods for Electroanalysis of the Catalytic Activity of Enzymes of Medical Significance

V.V. Shumyantseva1*, L.E. Agafonova1, T.V. Bulko1, P.I. Koroleva1, A.V. Kuzikov2, R.A. Masamrekh2, T.A. Filippova1

1Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail: viktoria.shumyantseva@ibmc.msk.ru

2Department of Biochemistry, Pirogov Russian National Research Medical University, 1 Ostrovitianova str., Moscow, 117997 Russia

Keywords: cytochrome P450; substrates; inhibitors; asparaginase; enzymatic catalysis; electrochemical analysis

DOI:10.18097/BMCRM00225

The whole version of this paper is available in Russian.

The review is devoted to new highly effective methods for analyzing the catalytic activity of enzymes of medical significance, such as cytochromes P450, trypsin, asparaginase, beta-lactamase, and nucleases. The methods are based on registration the specific activity of enzymes using electroanalytical methods. The review analyzes the experimental data obtained by the authors. Two platforms have been developed that allow quantitative measurement of catalytic activity based on the electrochemical properties of the enzyme (cytochrome P450, bactosomes, asparaginase) or substrate (trypsin, nucleases, restriction enzymes, beta-lactamase).
Figure 1. Immobilization of enzymes or substrates on electrodes to study catalytic activity.
Figure 2. Structure of the compound (2Ꞌ-{[(E)-3'-hydroxyandrost-5-en-17-ylidene]methyl}-4Ꞌ,5Ꞌ-dihydro-1Ꞌ,3Ꞌ-oxazole), which showed the greatest inhibitory activity towards cytochrome P450 17A1 in the 17α-hydroxylation reaction of pregnenolone [17, 18].
Figure 3. Two-electrode system for recording substrates or metabolites of cytochrome P450-dependent reactions. The principle of bielectrode electrochemical systems based on the use of an enzyme electrode with immobilized recombinant cytochrome P450 (or a membrane-bound form of this protein) and an indicator electrode on which the loss of substrate (A) or increase in product (B) is quantitatively determined by registration the electrochemical oxidation of these compounds during different potentials. As an example, an enzyme electrode with immobilized CYP2C9 is presented, which hydroxylates the marker substrate diclofenac with the formation of the reaction product 4ʹ-hydroxydiclofenac.
Figure 4. Methods for increasing the catalytic activity of cytochromes P450 in electrochemical systems.
Figure 5. Algorithm for electroanalysis of proteases based on the hydrolysis of peptides containing the electroactive amino acid tyrosine.
Figure 6. Differential pulse voltammetry of native plasmid DNA (-, 3 profile), DNA after exposure to specific restrictionase (-, on 4 fragments, 2 profile) and non-specific endonuclease (-, on 3-70 - mer fragments, 1 profile). As the degree of DNA fragmentation increases, the number of registered peaks corresponding to the electrooxidation of heterocyclic bases also increases, and the intensity of the maximum peak current for the electrochemical oxidation of heterocyclic bases increases.

CLOSE
Table 1. Determination of cytochrome P450 activity by electrochemical analysis of reaction products.

FUNDING

The work was performed within the framework of the Program for Basic Research in the Russian Federation for a long-term period (2021-2030) (№122030100168-2).

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