¹ Institute of Biomedical Chemistry, Moscow, Russia

² Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia

³ Foundation of Perspective Technologies and Novations, Moscow, Russia

⁴ Moscow State Academy of Veterinary Medicine and Biotechnology Named after Skryabin, Moscow, Russia

⁵ Faculty of Computational Mathematics and Cybernetics, Moscow State University, Moscow, Russia

The influence of an external constant strong electric field, formed using a pyramidal structure under a high electric potential, on an enzyme located near its apex, is studied. Horseradish peroxidase (HRP) is used as a model. In our experiments, a 27 kV direct current (DC) voltage was applied to two electrodes with a conducting pyramidal structure attached to one of them. The enzyme particles were visualized by atomic force microscopy (AFM) after the adsorption of the enzyme from its 0.1 µM solution onto mica AFM substrates. It is demonstrated that after the 40 min exposure to the electric field, the enzyme forms extended structures on mica, while in control experiments compact HRP particles are observed. After the exposure to the electric field, the majority of mica-adsorbed HRP particles had a height of 1.2 nm (as opposed to 1.0 nm in the case of control experiments), and the contribution of higher (>2.0 nm) particles was also considerable. This indicates the formation of high-order HRP aggregates under the influence of an applied electric field. At that, the enzymatic activity of HRP against its substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) remains unaffected. These results are important for studying macroscopic effects of strong electromagnetic fields on enzymes, as well as for the development of cellular structure models.

atomic force microscopy; horseradish peroxidase; protein aggregation; applied electric field

Electric fields play a crucial role in protein-protein and protein-lipid interactions (membrane processes), and in cell functioning. A large number of studies on the influence of electromagnetic [1,2,3,4,5] and magnetic [6,7,8] fields (both constant and alternating) on enzymes was reported, and in many of these works, horseradish peroxidase (HRP) is considered since it is a thoroughly studied model object [2,3,6,7,8,9,10,11,12]. For this enzyme, the effects of strong constant and alternating electromagnetic fields were studied. Zhang et al. [13] found that strong (30 kV/cm) pulsed electric fields have an inactivation effect on HRP, changing its conformation. Constant electric fields also affect HRP significantly. Among such papers, the study by Yang et al. on the influence of high-strength constant electric fields on the horseradish peroxidase activity [14] should be noted. These authors demonstrated an increase in the enzymatic activity of HRP at an electric field strength (E) in the range 2 × 10⁵ < E ≤ 6 × 10⁵ kV/cm, while no experiments were performed for stronger electric fields.

Two important points should be noted. Firstly, in a biological cell membrane, the electric field strength is about 2 × 10⁶ V/m. Secondly, these fields can be localized: for instance, as in protein pores of a funnel-shaped structure [15]. Thus, the influence of electric fields generated near model funnel-shaped structures, e.g., pyramidal or conical, on proteins and enzymes represents an actual task of modern biomedicine. Herein, a setup based on a pyramidal electrode has been used, and a solution of HRP enzyme was incubated near the apex of the pyramid. The strength of the electric field, generated with this element, was ~2 × 10⁶ to 10⁷ V/m, being similar to the one generated by a cell membrane. In addition, it should be mentioned that pyramidal structures can be used in biosensors [16].

Atomic force microscopy (AFM) was used as a research method since it allows visualization of single biological macromolecules [17,18]. AFM is a high-resolution technique, which is very useful in studying single polymer macromolecules [19]—in particular, proteins [18,20,21,22,23] and nucleic acids [24,25]—at a nanoscale. The height resolution of AFM is very high (~0.1 nm) and enables obtaining images of single biological macromolecules and their aggregates [9,10,11,12,21]. AFM was employed to study the influence of very weak electromagnetic fields on the aggregation state of HRP [9].

In the present work, HRP was used as a model enzyme, since it is characterized in much detail in the literature and has been used in studying the effect of electromagnetic fields on proteins [2,3,4,5,9,10,11,12,26]. HRP is a heme-containing enzyme glycoprotein with a molecular weight of 40–44 kDa [27,28], containing 18–27% structure-stabilizing carbohydrate residues [28,29]. In micromolar aqueous solutions, HRP tends to form aggregates [30]. At a concentration of ~10⁻⁷ M in a buffer solution, HRP exists as a mixture of monomers and aggregates [9].

Herein, by AFM, we have for the first time demonstrated that a 40 min exposure of 0.1 µM solution of HRP to a strong (27 kV) constant electric field applied to a pyramidal structure causes an increase in the enzyme aggregation on mica, which occurs in the form of extended structures. At the same time, its enzymatic activity remains unaffected. The results obtained are of great importance for the studying of enzyme and cellular structures, as well as for better understanding of the effect of strong electric fields on other enzyme systems.

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Ivanov, Y.D.; Tatur, V.Y.; Shumov, I.D.; Kozlov, A.F.; Valueva, A.A.; Ivanova, I.A.; Ershova, M.O.; Ivanova, N.D.; Stepanov, I.N.; Lukyanitsa, A.A.; Ziborov, V.S. Atomic Force Microscopy Study of the Effect of an Electric Field, Applied to a Pyramidal Structure, on Enzyme Biomolecules. J. Funct. Biomater. 2022, 13, 234. https://doi.org/10.3390/jfb13040234