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EFFECT OF MAGNETIC FIELDS ON LYSOZYME RENATURATION
R.M. Sarimov1*, T.A. Matveeva1, D.A. Serov1, P.I. Nikitin1, S.V. Bashkin2, A.A. Loboyko2, V.N. Binhi1
1 Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilov St., Moscow, 119991, Russia;
2 Bauman Moscow State Technical University, 5 Vtoraya Baumanskaya St., Moscow, 105005, Russia.
* Corresponding author: [email protected]
Abstract. Despite the large volume of empirical data on the effects of magnetic fields on living organisms, there are almost no studies showing the possibility of the existence of magnetic effects in processes outside the cell or in vitro. The protein folding process in a cell can be sensitive to magnetic fields. We the first time experimentally demonstrated the possibility of biochemical effects of extremely low frequency magnetic fields and hypomagnetic fields on chemical renaturation using hen egg-white lysozyme as an example. The degree of lysozyme renaturation was estimated at different magnetic conditions by fluorescence intensity and enzymatic activity. The extremely low frequency magnetic field (50 Hz, 40 ^T) accelerates the renaturation compared to the control and a hypomagnetic field (less than 40 nT). The effects of hypomagnetic and extremely low frequency magnetic field on the protein fluorescence spectrum were opposite. It confirms the participation of the recently described level mixing mechanism in the implementation of magnetobiological effects. The magnetic nanoparticles abolished the effects of extremely low frequency magnetic and hypomagnetic fields fluorescence and activity of lysozyme, which indicates their ability to modulate magnetobiological effects. The results obtained expand fundamental ideas about the mechanisms of action of magnetic fields on isolated protein molecules and can be useful in the practice of using magnetic nanoparticles in biomedicine.
Keywords: hen egg-white lysozyme, extremely low frequency magnetic field, hypomagnetic field, enzymatic activity, protein fluorescence.
List of Abbreviations
DLS — dynamic light scattering DTT — dithiothreitol
ELF-MF — extremely low frequency magnetic field
GdnHCL — guanidine hydrochloride GMF — geomagnetic field GSH — glutathione GSSG — glutathione disulfide HEWL — hen egg-white lysozyme HMF — hypomagnetic field IONP — iron oxide nanoparticles LMM — level mixing mechanism MF — magnetic fields NP — nanoparticles
TEM — transmission electron microscopy TRIS — tris(hydroxymethyl)aminomethane TSC — trisodium citrate
Introduction
At present, a large volume of experimental data has been accumulated on the influence of magnetic fields (MF) on living systems from the population to the molecular-cellular level of
organization (Belova & Acosta-Avalos, 2015; Kikuchi et al, 1998; Latypova et al., 2024; Maffei, 2022). However, the molecular mechanisms of most magnetobiological effects remain a mystery to this day. Biological effects of MF often occur already at low inductions, less than 1-2 p,T, when possible thermal effects are excluded (Alabdulgader et al., 2018; Mitsutake et al., 2004; Stepanova et al., 2015). In this case, the energy introduced into the system even by a high-frequency MF is many orders of magnitude less than the energy of thermal fluctuations of molecules. This leads to the so-called "kTproblem", expressed by the inequality mH«kT, where H is induction of the magnetic field, m is the magnetic moment of the supposed molecular target of the magnetic field, k is the Boltzmann constant, and T is the effective temperature of the target (Binhi & Prato, 2017; Lin et al., 1998). The search for mechanisms of the biological action of magnetic fields began at the end of the last century and remains an unsolved scientific problem to this day (Sarimov et al., 2023b). There are a
number of hypotheses describing the effect of magnetic fields on biological molecules (Sarimov et al., 2023a). Among the most probable and well-described mechanisms of magne-tobiological effects (Afanasyeva et al., 2006; Binhi, 2019; Binhi & Prato, 2017b; Buchachenko, 2014; Hore & Mouritsen, 2016; Liboff, 2019; Steiner & Ulrich, 2002). It is worth noting the mechanism of radical pairs (Hore & Mouritsen, 2016) and levels mixing mechanism (Binhi & Prato, 2017b). The participation of magnetic nanoparticles (NP) in the implementation of magnetobiological effects is also described in the literature, but remains a hypothesis. The indicated mechanisms of interaction of MF with rotating molecules and/or individual molecular groups are described in more detail in the work (Binhi & Prato, 2018).
Nanotechnology and nanobiotechnology are currently undergoing rapid development and are already successfully applied in a variety of fields: agriculture and food industry, wastewater treatment, environmental monitoring, biology and medicine, cancer therapy, and targeted drug delivery systems (Couto & Almeida, 2022; Gudkov et al., 2023; Khan et al., 2021; Santâs-Miguel et al., 2023; Semenova et al., 2024; Shurygina & Shurygin, 2018; Silant'ev et al., 2023). Due to the expansion of the areas of application of magnetic NPs, the mechanisms of interaction of NPs with eukaryotic cells are being actively studied. In this regard, the possible participation of NPs in cell responses to external MFs is of particular interest. Most of the studies devoted to magnetobiological effects have been performed at the level of organisms, starting with cells (Cortassa et al., 2003; Panagopoulos et al., 2002; Sarimov et al., 2023b; Sharpe et al., 2021). These studies provide a broad understanding of the possible biological consequences of external "man-made" extremely low frequency magnetic field (ELF-MF), magnetic storms, and/or removal of the geomagnetic field (hypomagnetic field, HMF). However, studies aimed at assessing the effect of MFs on individual protein molecules outside the cell are rare (Ogneva et al., 2020; Zhang et al., 2017). We believe that it is the assessment of the effect of MFs on individual
molecules that can be useful for clarifying knowledge about the mechanisms of magneto-biological effects.
An important aspect of protein functioning is the change in their conformation. In the most terminal cases, these changes are denaturation and renaturation. The available data on the effect of ELF-MF and HMF on protein folding are contradictory. On the one hand, there is data on the ability of ELF-MF and/or HMF to increase the probability of transcription and translation errors and prevent the acquisition of the optimal conformation of the protein molecule (Binhi, 2023; Sincák & Sedlakova-Kadukova, 2023). On the other hand, ELF-MF 8 Hz and 10 p,T increased the acetylation of HSP70 and HSP90 in AML12 and HEK293 cells (Huang et al., 2023). However, the cellular machinery is present in all these studies (Blackman et al., 1990; Blackman et al., 1989; Verheyen et al., 2003), which makes it difficult to isolate magnetically induced molecular and intracellular signaling events. We have not found any studies on the influence of ELF-MF and HMF on the processes of protein denaturation/renatura-tion in protein solutions without cells.
The study of fluorescence and enzymatic activity are simple, accessible and reproducible methods for assessing the conformational state and denaturation/renaturation processes of protein molecules (dos Santos Rodrigues et al., 2023; Nemukhin et al., 2009; Sarimov et al., 2021; Torgeson et al., 2022). Therefore, these methods were chosen in present work. The classic model protein for the study of fluorescence and enzymatic activity is hen egg-white lyso-zyme (HEWL) (Hirai et al., 2004; Khan et al., 2013; Summers & Flowers, 2008).
The aim of this work was to evaluate the effect of weak (with induction of the order of geomagnetic and less) MF on the process of renaturation/folding of proteins. To achieve this goal, we studied the enzymatic activity and fluorescence of HEWL in the following exposure modes: constant MF with induction of 40 p,T, ELF-MF with a frequency of 50 Hz and induction of 40 p,T, and under HMF (constant MF with induction less than 40 nT). Experimental data indicate that in some cases,
ELF-MF has a pronounced effect in certain frequency and amplitude "windows" (Baureus Koch et al., 2003; Krylov & Osipova, 2023; Sarimov et al., 2011; Sarimov et al., 2005). Many studies have shown that power transmission line frequencies (50-60 Hz), their harmonics and subharmonics cause magnetobiological effects (Astashev et al., 2023; Belova et al., 2010; Blackman et al., 1998; Ermakov et al., 2022). As an additional task, we studied the effect of the addition of magnetic NPs on the observed magnetic effects in relation to the specified protein characteristics. The influence of magnetic NPs is interesting in itself, since it is known that MFs near ferromagnetic NPs can exceed the geomagnetic field (GMF) by several orders of magnitude.
Materials and Methods
Materials and reagents
The work used egg white lysozyme EC 3.2.1.17 (hen egg-white lysozyme, HEWL >20,000 U/mg (Amresco, USA), as well as agents that affect the rate of protein denatura-tion and renaturation guanidine hydrochloride (GdnHCL), dithiothreitol (DTT), glutathione (GSH), glutathione disulfide (GSSG), triso-dium citrate (TSC), citric acid, tris(hydroxyme-thyl)aminomethane (TRIS), FeCh^^O, FeCh*6H2O and aqueous ammonia solution (all SigmaAldrich , USA). Lyophilized Micrococcus cells were used to assess the enzymatic activity lysodeikticus (ATCC No. 4698, Sigma-Aldrich, USA).
Synthesis of magnetic nanoparticles
Iron oxide nanoparticles (IONPs), both without modifications and coated with trisodium citrate (TSC-IONPs), were synthesised by the standard co-precipitation method of iron (II) and (III) chloride salt at alkaline conditions. Salts FeCl3*6H2O (5.9 g) and FeCh^O (2.15 g) were dissolved in 100 mL previously degassed MilliQ water. Further 12.5 mL of 30% NH4OH was quickly added. The obtained solution was heated to 85 °C and refluxed with vigorous stirring and N2 during 2 h. Further the formed IONPs were separated with NdFeB 5x5 cm magnet and washed one time with 2 M
HNO3 and three times with MilliQ water. The aggregates of NPs were then magnetically removed (Akopdzhanov et al., 2020; Nikitin et al., 2014; Sarimov et al., 2022). The trisodium citrate coated NPs (10 and 100 nm) was synthesized by method described previously (Akopdzhanov et al., 2018; Akopdzhanov et al., 2014; Brusentsov et al., 2012). To obtain TSC-IONPs 5 g of FeCl2 4H2O and 10 g FeCl3 6H2O were dissolved in 200 ml water, solution was heated at 70-80 °C. 25 ml in 25% NH4OH (added with vigorous stirring) during 40 min. IONPs were separated from sol with NdFeB magnet, washed out and diluted in 200 ml MilliQ water. Citric acid 12 g/l and TSC 44 g/l in 30 ml MilliQ water were added to IONPs sol and continuously stirred during 120 min at 7080 °C. TSC-IONPs were magnetically separated as described above. The stability and average size of the NP solution was also monitored using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The distribution of nanoparticles by size is shown in Figure 1. The initial concentration of TSC-IONPs 10 nm in the colloid after synthesis was ~1013 particles/ml. Concentrations for TSC-IONP 100 nm and IONP 350 nm were 3x1010 ml-1 and 6x109 ml-1 respectively. The nanopar-ticle morphology was investigated using a JEM-2100 (JEOL, Japan) transmission electron microscope operated at an acceleration voltage of 200 kV. Samples were prepared by dropping an aqueous dispersion NPs sol onto a carbon-coated copper grid or lacey carbon film grid followed by air drying. The uncoated IONPs formed aggregates during sample preparation. It making much more difficult to analyze the obtained TEM images, so no photographs of IONPs are presented. The IONPs were ultra-sonically shaken before the experiment and their size was approximately 350 nm in all experiments (Fig. 1, blue curve).
Chemical denaturation and renaturation of proteins
Chemical denaturation of HEWL 40 p,g/ml was performed according to the standard protocol in Tris-HCl (50 mM) buffer (pH 8.0) in the presence of 6 M GdnHCl and/or 30 mM DTT
Hydrodynamic diameter, nm A
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Fig. 1. Morphology of obtained NPs. Size distribution of NPs evaluated by DLS (A). Concentrations of TSC-IONP 10 nm, 100 nm and IONP 350 nm were 1013 ml-1, 3x1010 ml-1, and 6x109 ml-1 respectively. Mean ± SEM for 6 independent experiments. TEM microphotography of TSC-IONP with average size 100 nm (B) and 10 nm (C). Scale bars are presented on images
(Sarimov et al., 2021). In the control, the appropriate volume of Tris-HCl buffer was added to the protein solution instead of denaturing agents. Denaturation experiments were studied at a temperature of 24 ± 1 °C. To study renatur-ation dynamics, the denatured protein solution
was diluted 250-fold and a mixture of renatur-ing agents was added at final concentrations of 10 mM GSH and 2.5 mM GSSG. At selected time points, the optical density of protein solutions and their fluorescence were measured. HEWL enzymatic activity was studied accord-
Fig. 2. Magnetic exposure box for generating magnetic fields of the order of GMF and less (A) and exposure control system (B)
ing to the protocol described below. All protein renaturation processes were performed under different magnetic exposure conditions (see below).
Magnetic exposure
The magnetic exposure system developed by authors was used for experiment (Astashev et al., 2023). The magnetic exposure system is capable of creating a magnetic field with an induction of up to 100 and frequencies of up to 1 kHz to create controlled magnetic conditions in a volume of -10*10x10 cm. The system consists of three independent, orthogonally oriented Helmholtz pairs (Fig. 2A), a three-axis magnetic sensor FGM3D/100 (Sensys, Germany), a control unit, a USB-6343 ADC-DAC board (National Instruments, USA), a three-channel power amplifier and power supplies for it. The system was controlled using a PC and specially developed software (Fig. 2 B).
Solutions of the studied proteins in water or in the presence of selected denaturing/modifying agents were exposed to magnetic fields in different modes. In all cases, the magnetic exposure system suppressed random variations in the external magnetic field using feedback loop system. We used next modes:
1. Control was vertically directed static magnetic field (SMF) with induction of 40 p,T. All other components of the GMF are compensated.
2. ELF-MF with an induction of 40 and a frequency of 50 Hz (vertically orientated), against the background of compensated GMF with a residual induction of ~40 nT.
3. HMF is the regime with compensated GMF. Residual MF had ~40 nT induction with the maximum MF gradient (~20 nT/cm) at the location where the exposure system is installed.
Conformational changes in protein molecules were assessed using two methods: by the enzymatic activity of the protein using HEWL as an example and by the structure of the HEWL fluorescence spectra.
Fluorescence spectroscopy of lysozyme solutions
The fluorescence spectra of HEWL solutions were determined using a spectrofluorimeter. Jasco FP-8300 (JASCO Applied Sciences, Canada). The measurements were performed in quartz cuvettes with an optical path length of 10 mm. All parameters were selected so that the protein fluorescence peaks under control conditions corresponded to ~30% of the entire dy-
namic range of the device. At least three independent measurements were performed for each experimental variant. The measurements were performed at a constant temperature
(22 °C).
Determination of the enzymatic activity of lysozyme
The enzymatic activity of HEWL was assessed by the rate of bacterial cells Micrococcus lysodeikticus (ATCC 4698) lysis at room temperature. Bacteria were lysed in HEWL solution with a final concentration of 40 pg/mL in PBS (pH = 7.4) after the denaturation/renatura-tion procedure. Enzymatic activity was measured by the decreasing of optical density during the first two minutes after the addition of lyso-zyme with the Cintra 4040 spectrophotometer (GBC Scientific, Australia). The initial optical density at 450 nm was approximately ~0.8. The measurements were performed at 22 °C. The measurement technique has been described in detail in previous publications (Molkova et al., 2023; Sarimov et al., 2021). At least six independent measurements were performed for each experimental variant.
Statistical processing
The normality of distribution was assessed using the Kolmogorov-Smirnov test. The obtained data were presented as the mean value ± standard error of mean. The statistical significance of differences in sample mean values was assessed using the one- or two-way ANOVA with multiple comparisons by Dunnett 's method. In each experimental variant 3-6 independent measurements were performed. The exact sizes of the experimental samples are indicated individually in the corresponding figure captions.
Results
1. Effects of magnetic exposure on protein solutions without nanoparticles
Enzymatic activity during HEWL renaturation. Figure 3 shows the restoration of the hydrolyzing activity of HEWL during renaturation in three magnetic exposure modes.
The lysozyme activity restored faster under the influence of ELF-MF compare to the control (SMF). The HEWL activity is higher at all-time points (5, 15, 30, 60 minutes) except for the initial zero point. The maximum differences in activity were observed at the 30th minute. A statistically significant difference in activity was obtained under different exposure modes (ANOVA: Time (5 groups), Exposure (3 groups), Current effect: F(2,79) = 3.81, p = 0.026). For HMF in the interval of 15-30 min, an inflection of the "time-activity" line was observed.
Fluorescence during HEWL renaturation. Figure 4 shows the HEWL fluorescence spectra in the control and after 60 and 150 minute denaturation and reverse 60 minute renaturation. In the control, a fluorescence maximum was observed at 330-340 nm. This maximum was caused by the amino acids Trp, Tyr and Phe included in the protein. It's evident from the figure that the fluorescence intensity increases and the peak maximum shifts to the long-wave region during denaturation. The reverse processes occurred during renaturation. It is characteristic that denaturation continues even after 60 minutes, when the protein activity is almost completely absent (Fig. 4).
The change in the maximum fluorescence intensity during exposure in different modes is presented in the figures (Fig. 5 and Fig. 6). Exposure to ELF-MF also showed the maximum effects on the restoration of fluorescence intensity, while HMF showed the minimum effect (Fig. 5). Significant effects were recorded for the magnetic exposure modes (ANOVA: Time (5 groups), Exposure (3 groups), Current effect: F(2,28) = 3.52, p = 0.043). An interesting result was obtained in the restoration of the emission maximum wavelength. For all experimental variants, with increasing time in the range of 5-15 min, a shift to the low-frequency region occurred. After 14 min, a tendency to increase the emission maximum wavelength was observed for all experimental variants. The time-fluorescence curve for HMF has a characteristic inflection in the range of 15-30 min, which coincides with the inflection in the time-activity line during exposure to HMF (see above,
Fig. 3). Despite the fact that the tendency to shift the maxima on the time-fluorescence curves is not statistically significant, it is of
interest, since it may indicate the ability of HMF to change the activity of enzymes through a change in their conformation.
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Fig. 3. HEWL activity (40 |ig/ml during renaturation in 24 mM GdnHCL, 120 uM DTT , GSH 10 mM , GSSG 2.5 mM , TRIS 50 mM ) at HMF, ELF-MF 50 Hz 40 ¿iT or SMF 40 ¿iT (control). Mean ± SEM for 6-7 independent experiments. Two-Way ANOVA Factor exposure (SMF, ELF-MF, HMF) p < 0.026, *—p < 0.05 (Multiple Comparisons, Dunnett's Method): SMF 40 uT vs ELF-MF 50Hz 40 uTp < 0.021)
300 320 340 360 380 400 420 Emission, nm
Fig. 4. Fluorescence of HEWL (40 (jg/ml) at 60 and 150 min denaturation (red lines) in 6M GdnHCL, 30 mM solution DTT , 50 mM TRIS and reverse 60 min renaturation (blue lines) in a solution of 24 m M GdnHCL, 120 uM DTT, GSH 10 mM, GSSG 2.5 mM, TRIS 50 mM. The spectrum of control HEWL is shown as a black line
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Fig. 5. Fluorescence intensity at maximum emission upon excitation at 281 nm for HEWL (40 |ig/mL) during renaturation in 24 mM GdnHCL, 120 uM solution DTT, GSH 10 mM, GSSG 2.5 mM, TRIS 50 mM at HMF, ELF-MF 50 Hz 40 |iT or SMF 40 |iT (control). Mean ± SEM of 3 independent experiments. Two-Way ANOVA Factor Exposure (SMF, ELF-MF, HMF) p = 0.014
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Fig. 6. The maximum emission wavelength at excitation at 281 nm for HEWL (40 |ig/mL) during renaturation in a solution of 24 mM GdnHCL, 120 uM DTT, GSH 10 mM, GSSG 2.5 mM, TRIS 50 mM at HMF, ELF-MF 50 Hz 40 (j,T or SMF 40 |iT (control). Mean ± SEM for 3 independent experiments. No significant differences
2. Effects of magnetic exposure on protein renaturation in the presence of magnetic nanoparticles
In the present study, possible effects of changing the rate of renaturation in a solution with magnetic NPs under the influ-
ence of MF were also investigated. At this stage TSC-IONP 10 and 100 nm, and IONP 350 nm was investigated. Renaturation HEWL under the influence of MF was studied by activity and protein fluorescence.
HEWL renaturation -■- SMF 40 uT (Control) —•— HMF
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Enzymatic activity of HEWL in the presence of magnetic NPs. The figure (Fig. 7) shows the data of experiments where renaturation was studied by the activity of HEWL in different magnetic exposure modes with the addition of NPswith size of 350, 100 or 10 nm. The concentrations of TSC-IONP were selected in such a way that the activity of the renatured HEWL was not lower than 20% of the initial activity, which corresponds to the renatured protein without NPs (Fig. 8 A). TSC-IONP 100 nM alone at a concentration of 3^10 8 particles/mL increased HEWL activity after denaturation compared to the sample without NPs (Fig. 8 B). It can be seen from the figure that the addition of TSC-IONP of all studied sizes led to the
disappearance of the magnetic effects observed for HEWL in section 3.1.1 (Fig. 3). Studied TSC-IONP and IONP did not case significant differences in HEWL activity under all magnetic exposure modes (ANOVA, p > 0.05).
HEWL fluorescence in the presence of magnetic NPs. Similar results to the previous point were obtained for the study of HEWL renaturation by fluorescence (Fig. 9). It should be noted that TSC-IONP 100 nm strongly quenched HEWL fluorescence, presumably due to the formation of aggregates (Fig. 9 B). The addition of NPs not only abolished the effects of MF, but also significantly increased the dispersion of the experimental data.
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Fig. 7. HEWL activity (40 pg/ml) after addition of IONPs 350 nm 104 ml-1 (A), TSC-IONPs 100 nm 3.5x105 (B), or TSC-IONPs 10 nm 3x1012 ml-1 (C) during renaturation in 24 m M GdnHCL, 120 ^M solution DTT, GSH 10 mM, GSSG 2.5 mM, TRIS 50 mM at HMF, ELF-MF 50 Hz 4QxT or SMF 40 ^T (control). Mean ± SD for 3-4 independent experiments
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Fig. 8. Effects of TSC-IONP on the hydrolytic activity of lysozyme. (A) HEWL activity (40 ^g/mL) in native (orange), denatured (green) and renatured conditions after adding TSC-IONP of different sizes at selected concentrations. (B) Effect of concentration TSC-IONPs to the activity of denatured HEWL (10 ^g/ml). Mean ± SD of 3 independent experiments
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Fig. 9. Maximum intensity of HEWL fluorescence (40 ^g/mL) with addition of IONPs 350 nm 104 mL-1 (A), TSC-IONPs 100 nm 3.5x10 5 (B) or TSC-IONPs 10 nm 3x1012 mL-1 (C) during renaturation in 24 m M GdnHCL, 120 цМ solution DTT, GSH 10 mM, GSSG 2.5 mM, TRIS 50 mM at HMF, ELF-MF 50 Hz 40 цТ or SMF 40 цТ (control). Mean ± SD for 3 independent experiments. No significant differences
Discussion
The effects of ELF-MF and HMF on the processes of protein renaturation using HEWL as an example were studied in the present work. The effects were assessed by changes in the fluorescence spectrum and enzymatic activity of the protein.
The choice of exposure modes was determined by the following reasons. Laboratory MF is subject to uncontrolled random variations and its use in control could reduce the reproduc-ibility of the results obtained at different times. Therefore, a stabilized SMF with an induction of 40 p,T was used as a control exposure. This SMF is close to the GMF at the latitude of Moscow (~45 p,T). Exposure in a HMF was chosen based on theoretical assumptions (Binhi & Prato, 2017b), according to which the bioef-fects of an MF are more likely in a HMF. The biotropic effect of 50-Hz SMF has been repeatedly described in the literature, and the International Agency for Research on Cancer (IARC) placed ELF-MF with a frequency of 50-60 Hz and induction of > 0.3 p,T on the list of possible carcinogens (2B) (IARC, 2002).
Under our conditions, HEWL activity disappeared after 60 min of denaturation. However, according to the fluorescence data, we show (Fig. 4) that the protein after 60 min of denatur-ation does not differ much from the native protein and that denaturation continues. At the same time, it is much more difficult to renature the protein after 150 min of denaturation. Therefore, we used only the 60 min denatura-tion mode for magnetic exposure.
We have found that TSC-IONP 100 nm suppressed HEWL fluorescence. Works combining NPs and MFs are usually limited to studying the effects of magnetic hyperthermia. In this case, a decrease in the intensity of protein fluorescence was also observed. However, it was caused by thermal denaturation of proteins due to heating of magnetic NPs in MFs of relatively high frequency of hundreds of kHz and large amplitude of about three mT (Boitard etal., 2021). This is several orders of magnitude higher in frequency and induction than the MFs used in the present work. Thermal denaturation of the protein upon exposure to LF-EMF (50 Hz, 40 p,T) is unlikely in our experiments.
An acceleration of the restoration of the enzymatic activity of HEWL under the influence of ELF-MF was found (Fig. 3). Previously, other authors recorded an increase in the enzymatic activity of lactase under the influence of ELF-MF 40 Hz, which is partially agreement with our results (Wasak et al., 2019).
Theoretically, different mechanisms of change of catalytic activity of enzymes in the MF are possible (Binhi, 2011; Binhi & Rubin, 2023). The general concept of the action of the MF on organisms is the action through the change of dynamics of primary magnetic moments interacting with the MF. These can be spin magnetic moments of electrons (Hore & Mouritsen, 2016; Schulten et al., 1978) and nuclei (Binhi, 1995), moments of orbital motion (Binhi, 2019) and magnetic nanoparticles (Binhi & Chernavsi, 2005; Kirschvink et al., 2001).
The spin-chemical mechanism associated with the formation of intermediate spin-correlated pairs of radicals in the processes of electron intraprotein interaction is often discussed transport (Afanasyeva et al., 2006; Binhi, 2019; Buchachenko, 2014; Hore & Mouritsen, 2016; Schulten et al., 1978; Steiner & Ulrich, 2002). For example, in cryptochromes and photolyases due to photoexcitation cofactor FAD radical pairs FAD-Trp are formed. Some of these live long enough to produce magnetic effects. The HEWL lysozyme structure also contains two aromatic amino acid residues, phenylalanine 34 and tryptophan 109, near the active site (gluta-mine 35) (Held & van Smaalen, 2014) (Fig. 10). However, the active site does not contain a co-factor or coenzyme capable of initiating electron transfer and generating a radical pair. It is unclear whether radical pairs can be generated in any other way during the defolding/refolding of HEWL.
Another possible molecular mechanism of MF in proteins is the quantum level mixing mechanism (LMM) (Binhi & Prato, 2017b).
The primary sensors of weak MFs in this model are abstract magnetic moments of micro-particles: electrons, nuclei, atoms, molecules or molecular groups. This magnetic moments capable to precesse in MFs. Aromatic amino acids included in HEWL, in particular, tryptophans
Glutamine (E35)of active site
Fig. 10. Structure (PDB DOI: https://doi.org/ 10.2210/pdblRE2/pdb). Enlarged HEWL region near the active center (glutamine 35). Additionally, aromatic amino acids tryptophan 109 and phenylalanine 34 are highlighted
and phenylalanine are characterized by the presence of a large number of p-orbitals. These p-orbitals have a magnetic moment and are potential targets for HMFs and ELF-MFs (<300 Hz) MFs by the LMM mechanism. In a HMF, such targets will stop precessing, and in ELF-MFs they will precess unevenly (Binhi & Prato, 2017b), i.e., the effects of HMF and ELF-MFs on protein fluorescence would have a qualitative difference. We observed an increase in the HEWL fluorescence intensity in a HMF and a decrease in ELF-MF. The effects of ELF-MF and HMF on the renaturation of HEWL (activity and fluorescence) are not contrary to the predictions of LMM.
Magnetic NPs may be a possible target of ELF-MF in cells. Magnetite and maghemite NPs have been found in many organisms (Binhi, 1995). The calculated energy of a magnetic NP of -100 nm in size is many times greater than the activation energy kT of chemical reactions in a magnetic field of the order of GMF (Binhi & Chernavsi, 2005; Kirschvink et al, 2001). NPs fixed in tissues or the cytoskeleton can presumably deform nearby biological structures in ELF-MFs, which may lead to biological effects. In addition, magnetic NPs themselves create fairly strong MFs near their surface up to 100 mT at a distance of less than 100 nm
(Binhi, 2011). However, magnetic ef- fects are also observed in plant and animal cells deprived of NPs (Binhi & Prato, 2017). Consequently, there is currently no unambiguous confirmation or refutation of the participation of magnetic NPs in the nonspecific response of the organism to a magnetic field.
One of the aims of the present study was to experimentally test the "nanoparticle" hypothesis. Iron oxide nanoparticles are also interesting as they have antibacterial activity along with other metal oxides (Gudkov et al., 2024; Gudkov et al, 2023; Serov et al, 2024). The addition of TSC-IONPs had an effect on the enzymatic activity of lysozyme. First, the introduction of 100 nm TSC-IONPs increased the activity of lysozyme after denaturation. At a concentration of 3><108 particles/mL, the HEWL activity was restored almost twofold. Second, the addition of TSC-IONPs abolished the effect of the magnetic field on the enzymatic activity and fluorescence of HEWL. The obtained data indicate that magnetic NPs can play the role of modifiers of the responses of living systems to changes in the characteristics of the magnetic field. A case of enhancing the enzymatic activity of superoxide dismutase in the presence of magnetic IONPs (however, without surface coating) in a ELF-MF with an induction of more than mT and frequencies of
more than hundreds of kilohertz has been described in the literature (He et al., 2021). Moreover, with an increase in the size of the NPs and the induction of the ELF-MF, the catalytic activity of superoxide dismutase increased. The authors believed that the acceleration of the enzymatic reaction occurred due to possible local thermal effects. In our case, no significant increase in the enzymatic activity of HEWL is observed in the presence of TSC-IONPs at ELF-MF (50 Hz, 40 pT). It is apparently explained by the absence of thermal effects. Consequently, TSC-IONPs cause an additional non-thermal effect on the rate of protein renaturation in ELF-MF and HMF. Clarification and refinement of this mechanism is a task for future studies.
Conclusion
In this work, the effect of an extremely low frequency magnetic field with a frequency of 50 Hz and an induction of 40 pT and hypomag-netic field (<40 nT) on protein renaturation processes was investigated for the first time using hen white-egg lysozyme as an example. It was shown for the first time that ELF-MF accelares the restoration of enzymatic activi-
ty during renaturation compared to the control. It was found that HMF and ELF-MF acted differently on the catalytic activity of HEWL and the intensity of protein fluorescence during renaturation. The results obtained are consistent with theoretical concepts about the participation of molecular magnetic moments and LMM in magnetobiological effects on protein folding during renaturation. The effect of magnetic NPs on the enzymatic activity and fluorescence of HEWL was also found. TSC-IONPs dose-dependently increased HEWL activities after denaturation, but, at the same time, abolished the effects of AFM and HMF on renaturation HEWL, estimated by fluorescence spectra and enzymatic activity. The results obtained expand fundamental ideas about the mechanisms of action of magnetic fields on isolated protein molecules and can be useful when using magnetic NPs in biomedicine.
Funding
This research was funded by the Russian Science Foundation grant No. 22-22-00951, https://rscf.ru/en/project/22-22-00951/.
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