¹ Institute of Biomedical Chemistry, Moscow, Russia

² Foundation of Perspective Technologies and Novations, Moscow 115682, Russia

³ Moscow State University, Faculty of Computational Mathematics and Cybernetics, Moscow, Russia

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

⁵ Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow, Russia

Low-frequency electromagnetic fields, induced by alternating current, are known to influence physicochemical properties and functioning of enzymes, including their catalytic activity. Herein, by using atomic force microscopy (AFM) and spectrophotometry analysis in parallel, we have investigated how the incubation near an autotransformer operated at 50 Hz influences the physicochemical properties of horseradish peroxidase (HRP). We have found that 30 min incubation of the enzyme above the coil of a loaded autotransformer enhances a disaggregation of HRP on mica and the number of adsorbed enzyme particles by two orders of magnitude in comparison with the control sample. And most interestingly, the incubation of HRP above the switched-off transformer for the same period of time has been found to cause a disaggregation of the enzyme, An increase in the activity of HRP against ABTS has been observed in the 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.

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

Electricity has become a part and parcel of modern life, being ubiquitously employed both in industry and for household use. At that, alternating current (AC)‐based equipment is used most widely [1]. The main advantage of AC consists in the simple transformability of AC voltage [1]. This allows one to easily build high‐voltage AC lines and circuits, making the 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 low frequency range [2]. In the USA, 60 Hz commercial frequency is employed. 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 [2–7]. The typical “meeting point” of the AC equipment and the enzymes is a bioreactor with motor‐driven stirring device [8,9]. Of course, this is just the most demonstrative case, since the most common situation is the action of LFF on the AC equipment operator, and the processes in the human body are known to be regulated by enzymes [10]. The action of magnetic (and, hence, electromagnetic [1]) fields on an enzyme can well lead to a change in its spatial structure [11,12], and, accordingly, to the pathological state of the body [13]. Accordingly, the impact of external electromagnetic fields is one of the most important factors, whose effect on enzymes’ properties attracts attention of the modern science: impact of such fields on the body and, in particular, on enzymes was analyzed in many works [13–19]. 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 [4,11], and the variety of important enzymes is quite wide [10]. The evident effects of external fields, including LFFs, on enzymes [2–7] are thus forcing researchers to perform in‐deep studies of these phenomena.

In the literature, particular attention is paid to the effects of electromagnetic fields on horseradish peroxidase (HRP) enzyme [2–7,12], which has found numerous practical applications in both biotechnology and healthcare [20–27]. The functional properties of HRP were shown to change significantly under the action of electromagnetic fields [23], including LFFs [2,3,5]. Owing to the sensitivity of the adsorption/aggregation properties of HRP to the impacts of electromagnetic fields [6,7,28–30], this enzyme can be used as an electromagnetic radiation sensor [6,7,30]. In this connection, the sensitivity of methods employed for the detection of changes in the enzyme properties becomes a key point [28,29].

In studies of enzymes, various spectroscopy‐based methods are commonly employed [11,12,31,32]. These methods are, however, only helpful when the enzyme under study contains a chromophoric group (for instance, in case of cytochromes P450), or when changes in the enzyme’s spatial structure [11,12] or functional activity [2,3,11,29] are significant. At the same time, the changes in the enzyme’s properties are often quite subtle — though still important with regard to its functionality [29]. Such changes are barely distinguishable [11] or completely indistinguishable [28] by spectroscopic methods. If this is the case, atomic force microscopy (AFM) is quite helpful [6,7,28– 30]. AFM enables visualization of single enzyme molecules, thus allowing the scientists to reveal even subtle changes in the enzyme properties [6,7,28,29]. Parallel use of AFM and spectroscopic methods is also a good practice [28–30].

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

In order to find out how the incubation near the AC‐based equipment affects the HRP enzyme, the setup drawn in Figure 1 was employed. The setup included a standard LATR‐1 laboratory regulating autotransformer (Russia) and an Eppendorf 5810 R laboratory centrifuge (Eppendorf, Germany).

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Preprints ID: preprints-151512

Article title: Changes in Physicochemical Properties of HRP Induced by AC Electromagnetic Fields of Commercial Frequency

Doi: 10.20944/preprints202503.0577.v1

Website: https://www.preprints.org/manuscript/202503.0577/v1