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

⁵ Moscow State Academy of Veterinary Medicine and Biotechnology Named after Skryabin, Moscow, Russia

⁶ Department for Business Project Management, National Research Nuclear University “MEPhI”, Moscow, Russia

⁷ Institute for Urology and Reproductive Health, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia

⁸ Department of Infectious Diseases in Children, Faculty of Pediatrics, N.I. Pirogov Russian National Research Medical University, Moscow, Russia

Low-frequency electromagnetic fields, induced by alternating current (AC)-based equipment such as transformers, are known to influence the physicochemical properties and function of enzymes, including their catalytic activity. Herein, we have investigated how incubation near a 50 Hz AC autotransformer influences the physicochemical properties of horseradish peroxidase (HRP), by atomic force microscopy (AFM) and spectrophotometry. We found that a half-hour-long incubation of the enzyme above the coil of a loaded autotransformer promoted the adsorption of the monomeric form of HRP on mica, enhancing the number of adsorbed enzyme particles by two orders of magnitude in comparison with the control sample. Most interestingly, the incubation of HRP above the switched-off transformer, which was unplugged from the mains power supply, for the same period of time was also found to cause a disaggregation of the enzyme. Notably, an increase in the activity of HRP against ABTS was observed in both cases. We hope that the interesting effects reported will emphasize the importance of consideration of the influence of low-frequency electromagnetic fields on enzymes in the design of laboratory and industrial equipment intended for operation with enzyme systems. The effects revealed in our study indicate the importance of proper shielding of AC-based transformers in order to avoid the undesirable influence of low-frequency electromagnetic fields induced by these transformers on humans.

Keywords: low-frequency electromagnetic field; horseradish peroxidase; AC transformer; atomic force microscopy; enzymatic activity; enzyme disaggregation

Electricity has become a part and parcel of modern life, being ubiquitously employed both in industry and for household use. Currently, alternating current (AC)-based equipment is used most widely [1,2]. One main advantage of AC is the transformability of AC voltage [1]. This allows one to avoid heat loss by using high-voltage AC lines and circuits, thus making AC electric-power transmission preferable owing to its cost efficiency [1]. Accordingly, electric AC transformers represent key components of AC lines and equipment. In Europe, the commercial AC frequency is 50 Hz, pertaining to a low frequency range [1,2]. In Northern America, a 60 Hz commercial frequency is employed [1]. The operation of AC equipment, including AC transformers, is known to be accompanied by the induction of electromagnetic fields of respective frequency (low-frequency electromagnetic fields, LFFs). Low-frequency magnetic and electromagnetic fields are known to influence the physicochemical properties and functioning of enzymes [2,3,4,5,6,7]. Typically, the exposure of enzymes to AC equipment occurs in bioreactors with motor-driven stirring devices [8,9]. Of course, this is just the most illustrative case, since it is also common for LFFs to affect AC equipment operators, while the processes in the human body are known to be regulated by enzymes [10]. The impact of electromagnetic fields on the body and, in particular, on enzymes has been analyzed in many works [11,12,13,14,15,16,17]. Of course, the exact effect of an external field on an enzyme depends on the type of the enzyme and the parameters of the field [2,4,18], and the variety of important enzymes is quite wide [10]. The evident effects of external fields, including LFFs, on enzymes [2,3,4,5,6,7] thus motivate researchers to further study these phenomena.

In the literature, particular attention has been paid to the effects of magnetic and electromagnetic fields on the horseradish peroxidase (HRP) enzyme [2,3,4,5,6,7,11,18]. This enzyme has found numerous practical applications in biotechnology as a useful catalyst [19]. For instance, the uses of HRP for wastewater purification [20], in food technology [21] and in biofuel cells [22,23,24] have been reported. Furthermore, HRP is used in healthcare as a reporter enzyme in diagnostic systems [25,26]. This is why this enzyme attracts particular attention from scientists. The enzymatic activity of HRP was shown to change significantly under the action of electromagnetic fields [21], including LFFs [2,3,5]. Since LFFs are induced by various industrial AC-energized equipment (for instance, by transformers and electric motors) employed in biotechnological setups, a detailed investigation of their influence on the functionality of HRP is evidently required. Furthermore, the adsorption/aggregation properties of HRP were found to be quite sensitive to the influence of magnetic and electromagnetic fields [2,6,7,27,28,29]. Given the latter, this enzyme can be used as an electromagnetic radiation sensor [6,7,29]. To this end, the sensitivity of methods employed for the detection of changes in the enzyme’s properties has become a key point of study [27,28].

In studies of enzymes, various spectroscopy-based methods are commonly employed [11,18,30,31]. These methods are, however, only helpful when the enzyme under study contains a chromophoric group (for instance, in cases of cytochromes P450), or when changes in the enzyme’s spatial structure [11,18] and/or functional activity [2,3,18,28] are significant. A loss of activity often occurs due to denaturation [32]. Gajardo-Parra et al. [33] reported that the activity of HRP correlates with the α-helix content in its spatial structure. The denaturation of HRP can take place upon the action of chemical agents [32], pulsed light [34], high (70 °C and higher) temperatures [21,35] and microwave radiation [36]. Considering peroxidases in general, radio frequency [37] and microwave [38] radiation and various types of electric fields [39,40,41,42] were also reported to cause enzyme inactivation. This inactivation can also be ascribed to enzyme denaturation [40,41,42]. Indeed, stabilization of the spatial structure of HRP was shown to prevent its irreversible denaturation-caused inactivation [35]. The denaturation-caused inactivation of peroxidases can be unambiguously revealed by spectroscopy-based methods [34,35,37,39,40,41,42].

At the same time, the changes in the enzyme’s properties are often quite subtle, and, hence, are barely distinguishable [18] or completely indistinguishable [27] by spectroscopic methods. These changes can, nevertheless, be important with regard to enzyme functionality [28]. If this is the case, high-resolution methods are required in order to perform single-molecule investigations of the enzymes of interest. One well-known method for the high-resolution visualization of various objects of micron and sub-micron size is electron microscopy [43,44]. Transmission electron microscopy enables the visualization of studied specimens with sub-nanometer resolution, as was recently demonstrated for inorganic matter by Yang et al. [43]. Electron microscopy visualization of proteins, however, implies the use of harsh conditions (negative staining [45,46] or so-called vitreous ice [46]), which are far from native ones. In this respect, atomic force microscopy (AFM) is quite helpful [6,7,27,28,29]. Tapping-mode AFM enables the impact of AFM probes on the studied sample to be minimized upon the visualization of single enzyme molecules, providing their visualization under near-native conditions [47], thus allowing scientists to reveal even subtle changes in the enzyme properties [6,7,27,28]. The parallel use of AFM and spectroscopic methods is also a good practice [27,28,29].

In the work presented, the effect of incubation of the HRP solution near 50 Hz AC equipment on the enzyme’s physicochemical properties has been studied. It was observed that the incubation of the enzyme above the coil of a loaded autotransformer connected to a laboratory benchtop centrifuge led to the enhancement of HRP adsorption onto mica; this enhancement was accompanied by enzyme disaggregation and a slight increase in its activity. Furthermore, incubation near the transformer, which was switched off after its operation and disconnected from the mains power supply, was found to cause even more significant enzyme disaggregation, while the increase in activity was almost the same as in the case with the loaded transformer. Since 50 Hz AC-energized equipment is ubiquitously used in both research and industry, the results obtained in our experiments are quite important for the correct design of experimental procedures and industrial processes involving enzymes.

In our experiments, we used peroxidase from horseradish, which was purchased in the form of a commercial preparation from Sigma (St. Louis, MO, USA; Cat. #P6782; peroxidase from horseradish Type IV-A, essentially salt-free, lyophilized powder; batch No. SLCK8071; enzymatic activity against ABTS 1995 U/(mg solid), RZ 3.0 [48]). In addition, the enzyme was characterized by SDS-PAGE according to the technique reported by Ronzhina et al. [49] as described in the Supplementary Material. According to our data, the major fraction of the enzyme preparation had a molecular weight of 41 kDa (see Figure S1).

The 2,2^′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS; HRP substrate) was purchased from Sigma (St. Louis, MO, USA; Cat. #A1888). Disodium hydrogen orthophosphate, citric acid and hydrogen peroxide (H₂O₂) were purchased from Reakhim (Moscow, Russia). Dulbecco’s modified phosphate-buffered saline (PBSD) was prepared by dissolving a salt mixture, commercially available from Pierce (Appleton, WI, USA), in ultrapure water. All the solutions used in our experiments were prepared using ultrapure deionized water purified with a Simplicity UV system (Millipore, Molsheim, France).

In order to find out how the incubation near the AC-based equipment affected the HRP enzyme, the setup shown in Figure 1 was employed. The setup included a standard LATR-1 laboratory regulating autotransformer (Russia) and an Eppendorf 5810 R laboratory centrifuge (Eppendorf, Hamburg, Germany). This transformer was based on a toroidal magnetic circuit, which represented a ring-shaped electrical steel core with copper wire winding. The transformer was air-cooled, and rated for a nominal current of up to 9 A.

Figure 1. Schematic drawing of the experimental setup. The distance between the transformer’s coil and the test tube is 6 cm.

At the first step of the experiment, the autotransformer was loaded in the following way. The transformer’s input was connected to a 240 V, 50 Hz mains power supply. The output voltage was set to 220 V (Figure 1), and the centrifuge was connected to the transformer output. Then, the centrifuge was switched on and operated at 3000 rpm. The centrifuge power consumption was 1650 W. The centrifuge was located at a distance of 3 m from the transformer. The centrifuge had a metal body with plastic exterior and was grounded. We conducted experiments on the effect of the electromagnetic field of the centrifuge on the aggregation state of the enzyme and did not reveal its effect at a distance of 3 m from the centrifuge and 3 m from the transformer.

After switching the centrifuge on, a test tube with 1 mL of 0.1 µM HRP solution in 2 mM PBSD (the working sample) was placed onto a 8 mm thick textolite plate above the top of the transformer’s coil at a distance of 0.06 m, as shown in Figure 1. At the same time, the control sample was kept at a much larger (10 m) distance from the experimental setup.

The incubation time of the working sample above the loaded transformer upon the centrifuge operation was 30 min. In addition, one enzyme sample was placed into a grounded metal box (a Faraday cage) and incubated therein near the loaded transformer for the same period of time.

At the second step of our experiment, after 30 min of centrifuge operation, the centrifuge was stopped and switched off. The transformer was disconnected from the mains power supply. Five minutes later, another (untreated) working enzyme sample was placed in the same position above the transformer’s coil and incubated there for 30 min. At the points of incubation of the working and control enzyme samples under the conditions of our experiments, no temperature fluctuations exceeding 0.5 ^◦C were registered, as measured with an FY-10 thermocouple-based digital thermometer.

After the above-described procedures, all the enzyme samples studied were subjected to AFM analysis (in order to study the enzyme’s adsorption properties) and to spectrophotometric analysis (in order to determine the enzymatic activity). From each test tube with the studied enzyme samples, 200 µL of enzyme solution was taken for the analysis by spectrophotometry. The remaining 800 µL of each sample solution was used in the AFM analysis procedure described below.

The AFM and spectrophotometry analyses of the enzyme samples were performed in parallel as described in our previous papers—for instance, in [27–29]. The procedures, performed throughout these analyses, are briefly described in subsequent sections.

2.3. Atomic Force Microscopy Measurements

AFM analysis was performed by the direct surface adsorption method [47]. The adsorption was performed in Eppendorf-type test tubes. Each separate test tube contained an 800 µL volume of either of the samples studied, which were treated as described above in Section 2.2. One AFM substrate (7 mm × 15 mm sheet of freshly cleaved mica; SPI, Charlotte, NC, USA) was immersed into the analyzed enzyme sample solution in either of the test tubes, and incubated there for ten minutes. Each sample was analyzed in three technical replicates. The mica substrates were then scanned in semi-contact mode in air with a Titanium atomic force microscope (NT-MDT, Zelenograd, Russia; the microscope pertains to the equipment of the “Human Proteome” Core Facility of the Institute of Biomedical Chemistry, supported by the Ministry of Education and Science of Russian Federation, Agreement 14.621.21.0017, unique project ID: RFMEFI62117 × 0017). The microscope was equipped with NSG10 cantilevers (TipsNano, Zelenograd, Russia). For each substrate, at least sixteen scans (2 µm × 2 µm in size) were obtained in different areas of the substrate. Then, objects visualized in the so-obtained AFM images were counted with a specialized software custom-developed in IBMC. Based on the number of objects calculated on each AFM substrate (that is, for each enzyme sample studied), distributions of the relative number of objects with height ρ(h) (density functions) were obtained, and histograms of the absolute number of AFM-visualized particles N₄₀₀ (normalized per 400 µm²) were plotted vs. the height of the AFM-visualized objects [50]. The standard deviation in the AFM measurements was ≤10%, as obtained for three independent replicates measured for each sample.

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Ivanov, Y.D.; Shumov, I.D.; Kozlov, A.F.; Ableev, A.N.; Vinogradova, A.V.; Nevedrova, E.D.; Afonin, O.N.; Zhdanov, D.D.; Tatur, V.Y.; Lukyanitsa, A.A.; et al. Incubation of Horseradish Peroxidase near 50 Hz AC Equipment Promotes Its Disaggregation and Enzymatic Activity. Micromachines 2025, 16, 344. https://doi.org/10.3390/mi16030344