¹ Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, Russia

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

³ Foundation of Perspective Technologies and Novations, Russia

⁴ Faculty of Computational Mathematics and Cybernetics, Lomonosov Moscow State University, Russia

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

⁶ Associate Printing-and-Publication Centre Technosphera, Russia

⁷ Federal State Autonomous Educational Institution of Higher Education «Bauman Moscow State Technical University», Russia

⁸ Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Republic of Belarus

Heme-containing enzymes play vital functions in living organisms, including humans. Here we demonstrate two indirect effects of (electric discharge)-treated stainless steel on a model enzyme — horseradish peroxidase (HRP). The first effect is the complete loss of the enzyme’s adsorption after its incubation in grounded stainless steel chamber, which has been preliminarily subjected to electric discharge in air at atmospheric pressure. The second one is the formation of enzyme aggregates in the sample incubated in another grounded chamber two meters away from the discharge-treated one. At that, the HRP’s enzymatic activity is found to be unaffected in the both cases. These effects may be explained by the occurrence of knotted electromagnetic fields (KEMF). By using high-speed atomic force microscopy (HS-AFM), we reveal the relatively high surface mobility of cytochromes P450cam and P450 102A1 (BM3), whose isoelectric point (pI) values are acidic; at that, thymidylate synthase (TYMS) with near-neutral pI adsorbs strongly. Thus, HRP is the best model object, since its basic pI provides quite strong adsorption on mica. Since (electric discharge)-processed materials have found applications in medicine, we expect that the effects discovered will be considered in future biomedical applications of (electric discharge)-based technologies.

Keywords: electric discharge; atomic force microscopy; peroxidase; cytochrome P450; enzyme aggregation; enzyme adsorption; knotted electromagnetic field

Gas discharges occur upon passing of electric current through a gas at various gas pressures ranging from atmospheric one down to several Pascales [1–3]; at that, the gas turns into either partially or fully ionized state, which has been called plasma [1]. To date, both the gas discharge as a phenomenon and the plasma (produced from the initial plasma-forming gas) as a medium have found numerous technological applications. The applications of gas-discharge plasma include – but are not limited to - water purification [4,5], surface decontamination [6] and sterilization [7,8]. Gas discharges, in their turn, are applied, for instance, for precise machining of conductive materials (metals and alloys) [9], including steel [10,11]. Regarding the la er, the use of electric discharge machining (EDM) for fabrication of medical tools should be emphasized ^9,12. Technologies based on gas discharges have also found many other applications in medicine, including surface treatment of both inorganic and organic materials [13].

Interaction of an electric discharge with biological macromolecules can be either direct or indirect (Figure 1a) [14]. The direct interaction implies direct contact of the gas-discharge plasma with the biological macromolecules. In contrast, an interaction of discharge-processed materials with the biological macromolecules without their direct introduction into the discharge area represents the indirect interaction [15,16]. While direct effects of electric discharge on biological macromolecules were already reported [17,18], the indirect ones are poorly studied. Typical example of the la er is the dissolution of a sample in plasma-processed water [16].

(a)

(b)

Figure 1. Schematic illustration of direct and indirect action of electric discharge on an enzyme sample (a), and implementation of the indirect action of electric discharge in our experiments (b, not to scale).

Direct effects of gas discharges and gas-discharge plasma on biomolecules, including enzymes, are well known [17,18], though research in this direction is actively continued. In contrast, as we are aware, indirect effects of (electric discharge)-processed solid materials on biomolecules are yet unknown. At that, electric discharges are used for processing of conductive materials (including steel [10,11] intended for medical applications [9,12], thus implying indirect interaction of electric discharge on biological tissues and macromolecules — including enzymes. The la er is what we consider herein.

Atomic force microscopy (AFM) was shown to be a useful approach to single-molecule imaging of heme-containing enzyme [19,20]. AFM allows one to reveal even minor effects of magnetic [21,22] and electromagnetic [23,24] fields on their physicochemical properties. Heme-containing enzymes catalyze many vital processes in living organisms. In general, high-valent iron-oxo heme proteins catalyze oxidation of various compounds by either molecular oxygen or hydrogen peroxide [25]. Namely, monooxygenases of cytochrome P450 superfamily play key roles in oxygenation of fa y acids and various xenobiotics by molecular oxygen [26,27]. Heme-containing peroxidases, in their turn, participate in oxidation of various compounds by hydrogen peroxide [28]. The key roles of the processes, catalyzed by heme-containing enzymes, determines the importance of studies concerning the influence of various external factors on the functioning of heme-containing enzymes. These factors include (though are not limited to) [29,30] electromagnetic fields, which may affect enzyme structure and functionality [23,31,32].

In order to justify the choice of HRP as a model enzyme in experiments on the investigation of indirect effects of (electric discharge)-treated stainless steel on heme-containing enzymes, we use the illustrative examples of enzymes with acidic and near-neutral isoelectric point (pI) values. The data obtained for HRP are compared with the effect caused by direct exposure of the enzyme to 10 nW/cm² knotted electromagnetic field (KEMF). The first effect of the discharge-processed steel is the complete loss of the enzyme’s adsorbability after its incubation in grounded stainless steel container, which has been preliminarily subjected to electric discharge in air at atmospheric pressure. The second effect is the formation of enzyme aggregates in the sample incubated in another grounded container two meters away from the discharge-treated one. At that, the HRP’s enzymatic activity is found to be unaffected in the both cases. Direct exposure to KEMF is shown to induce considerable aggregation of the enzyme, do not affecting its activity. These effects may be explained by the occurrence of KEMF [23]. Since (electric discharge)-processed materials find their application in medicine [13], we expect that the effects discovered herein will be considered in future biomedical applications of (electric discharge)-based technologies.

High-speed atomic force microscopy (HS-AFM) developed by Professor T. Ando et al. [33,34] represents a powerful tool, which allows one to visualize dynamic processes in enzyme systems (including heme-containing enzymes) [35] with high time resolution. By HS-AFM, Takeda et al. demonstrated real-time visualization of adsorption of cytochrome C [35]. Continuous successful work on improving the spatial resolution and reducing noise of HS-AFM imaging is being performed, as was quite recently demonstrated by Sato et al. [36]. Herein, by HS-AFM, we experimentally demonstrate high surface mobility of two different cytochromes of P450 superfamily — P450cam and cytochrome P450 102A1 (P450 BM3) — in buffer with physiological pH 7.4; the isoelectric point (pI) values of these enzymes are acidic [37,38]. This is in contrast to the case with thymidylate synthase (TYMS), whose pI is near-neutral [39]. This is how we motivate the choice of horseradish peroxidase (HRP) with basic pI as a model enzyme in the investigation of effect of electric discharge-treated steel on a heme-containing enzyme in subsequent experiments reported herein.

формате PDF (1905Кб)