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.

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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