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 The All-Russian Research Institute for Optical and Physical Measurements Federal State Unitary Enterprise (VNIIOFI) of the Federal Agency for Technical Regulation and Metrology of the Russian Federation, Moscow, Russia
Our present study concerns the influence of the picosecond rise-time-pulsed electromagnetic field, and the impact of nanosecond pulsed pressure on the aggregation state of horseradish peroxidase (HRP) as a model enzyme. The influence of a 640 kV/m pulsed electromagnetic field with a pulse rise-time of ~200 ps on the activity and aggregation state of an enzyme is studied by the single-molecule atomic force microscopy (AFM) method. The influence of such a field is shown to lead to aggregation of the protein and to a decrease in its enzymatic activity. Moreover, the effect of a shock wave with a pressure front rise-time of 80 ns on the increase in the HRP aggregation is demonstrated. The results obtained herein can be of use in modeling the impact of electromagnetic and pressure pulses on enzymes and on whole living organisms. Our results are also important for taking into account the effect of pulsed fields on the body in the development of drugs, therapeutic procedures, and novel highly sensitive medical diagnosticums.
Keywords: horseradish peroxidase; atomic force microscopy; protein aggregation; shock wave; pulsed electromagnetic field
Biological systems are often subjected to external influences. Electromagnetic fields are now actively used in both industry and everyday life . Moreover, the use of microwave electromagnetic radiation for medical applications is discussed [2,3,4,5]. High pressure is another type of external impact, which also takes place in nature, and can thus affect protein structure . Such external influences are known to be able to affect the protein structure [6,7]—including the aggregation state—and other physicochemical properties of proteins. These properties of proteins, in their turn, determine the proper functioning of the entire organism. This is why studying the effect of electromagnetic and pressure fields on proteins represents an actual problem of modern life science.
Regarding the aggregation state of proteins, in animals and humans, protein aggregates can have either positive or negative effects. The positive effect consists of providing proper functioning of the body; for instance, myeloperoxidase functions in dimeric form , formed by functionally independent monomeric units joined by a single disulfide bond at Cys153 . Another example is glutathione peroxidase, which participates in oxidative stress regulation and is functionally active in tetrameric form .
The negative effect of protein aggregation manifests itself in the form of the development of various pathologies in the body caused by the formation of protein aggregates. Namely, protein aggregation is known to be a cause of cardiovascular diseases . Furthermore, protein aggregation was reported to be associated with cancer in humans: Xu et al.  reported that aggregation processes, which involve mutant p53 protein, are associated with the development of cancer, which could thus be considered as aggregation-associated disease. This was confirmed in the research performed by other authors [13,14,15]. Another well-known fact is that the formation of amyloid aggregates in the brain was considered to lead to Parkinson’s  and Alzheimer’s [16,17,18] diseases. In this connection, it should be, however, emphasized that certain proteins in amyloid form are relevant for proper brain functioning: therefore, in mammals’ brain, FXR1 protein, which regulates memory and emotions, is functioning in amyloid form .
Medicinal agents, intended for the treatment of diseases caused by protein aggregation, are often non-specifically disrupting the protein aggregates. During such a non-specific therapy, however, not only the target pathology-associated protein aggregates but also the ones required for proper body functioning, can be destroyed. In this connection, the revelation and investigation of factors influencing protein aggregation, the development of methods for the assessment of this influence, and the development of novel approaches providing specific correction of protein aggregation, represent important tasks of modern biomedical research aimed at maintaining human health.
Regarding magnetic (MFs) and electromagnetic fields (EMFs), a lot of attention is given to these fields with relatively long rise-times. Both extremely low frequency (20 to 75 Hz [1,19,20,21]) and microwave frequency EMFs [22,23,24] are known to influence protein structure and/or functionality. Regarding protein structure changes, irradiation in 940 MHz circularly polarized EMF was shown to cause partial unfolding of adult hemoglobin . Lopes et al.  reported that 0.5 h-long microwave irradiation (at a temperature of 60 °C and radiation power of 60 W) of horseradish peroxidase leads to significant (~80%) loss in its enzymatic activity. Moreover, Latorre et al. have demonstrated that just 30 s irradiation of a sample containing red beet peroxidase and polyphenoloxidase in 2450 GHz EMF (at 450 W microwave power) causes a very significant decrease in their enzymatic activity: namely, these authors observed 15-fold and 100-fold decrease in the activity of red beet peroxidase and polyphenoloxidase, respectively . The influence of EMF with extremely low frequency (ELF-EMF) can either stimulate or suppress the activity of enzymes. Therefore, Morelli et al. found that a number of membrane-associated enzymes (alkaline phosphatase, acetylcholinesterase from blood cell membranes, acetylcholinesterase from synaptosomes, phosphoglycerate kinase, and adenylate kinase) lose their activity upon the influence of a 75 Hz ELF-EMF, while other enzymes (CaATPase, Na/K ATPase, and succinic dehydrogenase) are virtually insensitive to such an influence . Thumm et al. observed a 2-fold increase in the activity of cAMP-dependent protein kinase in human skin fibroblasts after 1 h exposure to 20 Hz ELF-EMF (7–8 mT) . Wasak et al. demonstrated that the enzymatic activity of horseradish peroxidase (HRP) can either increase or decrease after its exposure to an extremely low frequency (1 to 50 Hz) rotating MF, depending on the MF parameters . Caliga et al.  observed a ~2-fold decrease in the HRP catalytic efficiency after its incubation in a 50 Hz (2.7 mT) ELF-EMF, while a 100 Hz (5.5 mT) ELF-EMF had virtually no effect on the enzyme. Modulation of the activity of enzymes by MFs can be explained by the interaction of the MF with the 3D structure of the protein . The number of papers reporting the effects of electric and/or electromagnetic fields with picosecond rise-times on biological systems is quite low. Considering the cellular level, Gao et al.  reported that an extremely high intensity pulsed electric field with 150 ps rise-time is able to permeabilize biological cells. In the works considering the influence of pulsed electromagnetic fields at the protein level, the studied range of pulse rise-times is limited to nanoseconds . Application of pulsed electromagnetic fields (PEMF) with nanosecond rise-times was demonstrated to enhance the sensitivity of diagnostic systems . Moreover, the possibility of the use of non-thermal effects of low-power (from 10−5 to 10−3 W/cm2) sub-nanosecond pulsed radiation (with a rise-time of 200 ps) for cancer therapy was reported . Studies on the application of microwave imaging for cancer diagnosis are also reported [2,4,5]. In this connection, studying the effect of pulsed non-thermal electromagnetic radiation on enzymes represents an important task for both fundamental and applied research, including medical and diagnostic applications . Sinusoidal electromagnetic fields of GHz frequency were reported to influence the activity of heme-containing enzymes . We are, however, not aware of any studies reporting such an influence from pulsed electromagnetic fields with shorter (picosecond) rise-times; accordingly, additional research is required to investigate whether such fields have an influence on enzymes.
Regarding pressure fields, they are also known to influence the structure and functionality of proteins  and enzymes [6,30,31,32,33]. Similar to electromagnetic radiation, the mechanical pressure, acting on biological objects, can be constant (hydrostatic) [6,30] or pulsed. The effects of hydrostatic pressure on enzyme systems were extensively studied previously. So, G. Hui Bon Hoa et al.  studied the impact of a constant 1000 to 3000 bar pressure on the properties of heme-containing enzymes and demonstrated that such an impact can lead to a change in their structure and functional activity. The latter case is very interesting with regard to studying electron transport systems, which participate in the metabolism of various compounds in the body . In their review, Eisenmerger and Reyes-De-Corcuera  discuss the effect of high (up to hundreds MPa) hydrostatic pressure on various types of enzymes (oxidoreductases, transferases, hydrolases, and lyases). These authors emphasize that in general, high pressure can help to stabilize enzymes against thermal denaturation, but at the same time, the enzymatic activity can be either increased or suppressed depending on each specific case. Andreou et al.  and Akazawa et al.  demonstrated that the sensitivity to high pressure is the individual characteristic of each enzyme. In experiments performed by Andreou et al., the enzymatic activity of polygalacturonase decreased rapidly (down to complete inactivation after 10 min treatment) upon the action of high (up to 500 MPa) pressure, while pectinmethylesterase retained ~70% of its initial enzymatic activity even after 20 min treatment at 800 MPa . Akazawa et al. observed a decrease in β-glucosidase and lipase activities at 0.1 MPa and higher pressures, while α-amylase exhibited higher activity at high (400 MPa) pressure; these authors also demonstrated pressure-induced activation of lumbrokinase at 200 MPa and higher (up to 500 MPa) pressures . Once again, in the above-mentioned papers [6,30], studies of the pressure impact on proteins are limited to the cases with constant pressure. In this regard, studying the influence of fast rise-time-pulsed pressure impacts on proteins represents an actual direction of research.
Peroxidase enzyme systems are known to perform important functions in various metabolic processes. Peroxidases pertain to heme-containing enzymes, which are well represented in plant and animal tissues . This provokes a great interest in studying this class of enzymes. In humans, myeloperoxidase participates in atherogenesis  and in oxidative stress . For these considerations, a peroxidase has been employed in our present study as a model object. Namely, horseradish peroxidase, which is comprehensively characterized in the literature, is often employed as a model object in studies of a wide class of peroxidases. HRP represents a glycoprotein with a molecular weight of about 40 to 44 kDa [35,36], which can form aggregates . HRP catalyzes the oxidation of many organic and inorganic compounds by hydrogen peroxide . It is known that HRP represents a D-isomer, whose structure includes 77% α-helices and 12% β-sheets ; its macromolecule also includes 18% to 27% of carbohydrate chains, which stabilize the protein structure [36,40,41]. Accordingly, the structure of this enzyme is chiral (or more exactly, pseudo-chiral).
AFM allows one to visualize and measure the functional activity of single enzyme molecules [42,43], thus representing a very convenient tool for the determination of their aggregation state. This is quite useful for single-molecule enzymology. For this reason, in our research, atomic force microscopy (AFM) was employed to study the effect of external factors on HRP aggregation. Previously, AFM was employed to study the formation of various protein complexes [44,45,46,47,48,49,50,51,52,53]. In parallel, in our present study, a commonly used spectrophotometry-based technique was employed to estimate the enzymatic activity of HRP in solution.
Our present work is aimed at the AFM investigation of the influence of short (sub-nanosecond and nanosecond) electromagnetic (640 kV/m) and pressure (10 atm) pulses on protein aggregation. The pulse rise-time was ~200 ps in the case of PEMF, and 80 ns in the case of the pulsed pressure field. In the literature, such pulses were called ultra-short electromagnetic pulses (USEMP) . The parameters of the electromagnetic field have been chosen based on the above-discussed factors, since such electromagnetic field characteristics are interesting from a practical point of view for diagnostic applications (for instance, for microwave imaging of cancer [4,5]). Accordingly, as discussed above, it is important to find out whether electromagnetic fields with such characteristics have an effect on enzymes. And herein we have studied the influence of PEMF using HRP as a model enzyme.
It has been demonstrated that an increased aggregation of the HRP molecules is observed after the exposure of its solution to PEMF. Moreover, the enzymatic activity of HRP also decreased considerably after the irradiation of the enzyme solution in PEMF. We have also demonstrated that a pressure jump, induced by the action of a fast rise-time (of the order of 80 ns) shock wave, leads to an aggregation of the protein. The influence of the pressure jump and the pulsed electromagnetic field on the protein aggregation state is discussed. It has been considered that, according to the literature , protein aggregation can lead to a change in circular dichroism spectra, i.e., influences stereochemical properties of the protein molecules. This aspect is interesting due to the fact that chirality and specific stereochemical properties of biological molecules are commonly occurring in living organisms, however, the nature of this phenomenon is still unknown.
The results obtained herein can find their application in the development of models describing the interaction of electromagnetic and pressure fields with enzyme systems and with whole organisms. The data obtained can also be used in further studies aimed at the development of safety standards regarding the practical use of electromagnetic fields. Our results should also be taken into account in the development of novel highly sensitive diagnosticums, intended for the revelation of diseases associated with protein aggregation, and new medicinal agents intended for the treatment of these pathologies.
Appl. Sci. 2021, 11, 11677
Ziborov, V.S.; Pleshakova, T.O.; Shumov, I.D.; Kozlov, A.F.; Valueva, A.A.; Ivanova, I.A.; Ershova, M.O.; Larionov, D.I.; Evdokimov, A.N.; Tatur, V.Y.; Aleshko, A.I.; Sakharov, K.Y.; Dolgoborodov, A.Y.; Fortov, V.E.; Archakov, A.I.; Ivanov, Y.D. The Impact of Fast-Rise-Time Electromagnetic Field and Pressure on the Aggregation of Peroxidase upon Its Adsorption onto Mica. Appl. Sci. 2021, 11, 11677. https://doi.org/10.3390/app112411677