ELF-EMF exposure decreases the peroxidase catalytic efficiency in vitro

Răzvan Caliga; Călin Lucian Maniu; Marius Mihăşan

doi:10.1515/biol-2016-0009

Article Open Access

ELF-EMF exposure decreases the peroxidase catalytic efficiency in vitro

Răzvan Caliga , Călin Lucian Maniu and Marius Mihăşan

Published/Copyright: May 6, 2016

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From the journal Open Life Sciences Volume 11 Issue 1

Abstract

The activity of purified Fe³⁺ containing horseradish peroxidase (HRP) was recorded while the enzyme was exposed to two low frequency pulsed electromagnetic fields (ELF-EMFs) with different frequencies: 50 Hz/2.7 mT and 100 Hz/5.5 mT. No statistically significant difference in the Michaelis constant (K_m) values between the control enzyme and the enzyme exposed to any of the two ELF-EMF used could be detected (53 ± 11 μM vs 67 ± 10 μM vs 42 ± 11 μM respectively), indicating that upon short exposure times, there are no large structural alterations of the enzyme that affect the substrate affinity. A 50 Hz/2.7 mT EMF did however, cause a significant decrease in the maximum rate (V_max) value (from 3.31 ± 0.17 μmoles/min to 1.6 ± 0.19 μmoles/min, p<0.03) and a drop of the catalytic efficiency to less than half (from 0.66 ± 0.03 s^-1 to 0.32 ± 0.04 s^-1, p<0.05). These observations are the first experimental proofs that support the previously postulated mechanism of coupling between ELF-EMF and living systems based on a resonant frequency specific for a given ion molecule.

Keywords: Fe3+ containing horseradish peroxidase (HRP); kinetics; catalytic efficiency; extremely low frequency electromagnetic field (ELF-EMF)

1 Introduction

The interaction of different types of electromagnetic fields (EMF) with living matter is an area of research that has emerged with the dawn of electricity and magnetism. There is now substantial experimental evidence that EMFs influence many cellular and metabolic activities. A plethora of papers deal with the effect of EMFs on immunity [1-2], kinetics of cell proliferation [3], gene expression [4], protein synthesis and folding [5], DNA damage [6], transmembrane ionic fluxes [7] and on enzyme activities. The influence of EMFs on enzymes and on the enzymatic catalytic mechanisms are of particular interest because of the central role that enzymatic proteins play in biological processes. The relation between EMFs exposure and the catalytic activity have been studied for several key enzymes, such as Na⁺-K⁺ ATPase [8], catalase [9], peroxidase [10], superoxide-dismutase [11], cytochrome oxidase [12], cellulase [13], ornithine decarboxylase [14], adenylate kinase [15], amylase [16], calpain [17] and ascorbic acid oxidase [18]. All of the experiments have used long exposure times (up to 48 h) and dealt mainly with the thermal effect of the radiation or with the structural changes induced by the EMFs [19]. From the many studies carried out, some general conclusions can be inferred. First, the interaction of EMF with biological systems (enzymes included) is closely correlated with the applied frequency. In case of the extremely low frequency (0-300 Hz) electromagnetic fields (ELF-EMF), the mechanism of interaction is slightly different when compared with the medium and high frequency EMFs. At low frequencies, electric and magnetic components of an electromagnetic field act independently: there is no “true” electromagnetic field in comparison to at much higher frequencies [20]. The way in which the ELF-EMF components act on a biological probe should not be interpreted in the same terms as high frequency fields as it may lead to erroneous conclusions [21].

Though significant progress has been made towards accurately describing the ELF-EMFs mode of interaction with enzymes, the issue remains controversial [22]. In an attempt to obtain further insights in this direction as well as on the ELF-EMFs influence on catalytic mechanisms of enzymes, we have analysed the effect of an ELF-EMF on pure preparations of the well-known enzyme horseradish peroxidase (HRP, phenolic donor: hydrogen-peroxide oxidoreductase, E.C. 1.11.1.17) [23]. The rationale for the choice of this particular enzyme is based on several key points. It has been shown previously that even weak magnetic fields applied to living organisms produce variations in the free-ions concentrations within the cells [11]. HRP contains two different types of metal ions, iron (III, in the heme group) and two calcium atoms, all of which are essential for the structural and functional integrity of the enzyme [24]. One might expect thereby that ELFEMFs exposure would alter the HRP catalytic properties. Indirect evidence that this might be the case exist, as HRP is one of the main cellular defence systems against different reactive oxygen species (ROS) and the literature pool is rich in reports indicating that ELF-EMF exposure induces oxidative damage in living cells [2,11,25,26]. Are these changes the direct effect of the ELF-EMFs ability to produce free-radicals [27]? Or, are the EMFs interacting with the key players of the anti-oxidant cellular systems, the metal-containing enzymes superoxide-dismutase, peroxidase and catalase?

Most of the studies performed so far follow the same experimental scheme: the purified protein (free or immobilised) was exposed for long periods of time to ELF-EMF and the effect was evaluated by comparing the enzyme activity prior and after exposure. We report here the results of a different approach, which attempts to evaluate the effects of ELF-EMF not only on the enzyme itself, but also on the catalytic reaction. For these experiments, the exposure time was reduced to 5 min and the activity of HRP was recorded while the enzyme was under the influence of ELF-EMF. The kinetic parameters were calculated for the enzyme before and during exposure.

2 Material and Methods

2.1 Chemicals and biochemicals

All chemicals were of the highest purity available (Na₂HPO₄, NaH₂PO₄, and H₂SO₄ were analytical grade, H₂O₂ was ACS grade and o-dianisidine was spectrophotometric grade) and were purchased from Sigma-Aldrich (Steinheim, Germany). Sigma Type XII Peroxidase from horseradish was obtained also from Sigma-Aldrich and had an indicated specific activity of 250–330 units/mg solid.

2.2 ELF-EMF exposure system

The device used for HRP exposure to EMF generates a pulsed electromagnetic field through two Helmholtz coils. The pulsed DC generator was constructed using the electronic scheme depicted in Figure 1, A. When using a pulsed DC with a frequency of 50 Hz or 100 Hz, the peak voltage DC variation applied to each coil is 42 V as shown on the oscilloscope (model 5115, Tektronix, Köln, Germany), (Figure 1, B and C) and with an intensity of 0.7 A at 50 Hz and 1.4 A at 100 Hz as indicated by an ammeter (model PA-212, Che Scientific Co., Hong-Kong, China). The coils have a diameter of 29 cm and approximately 630 turns. Magnetic flux density values on the central axis were measured with a multifunctional teslameter (model PG-5A, Yuxiang Magnetic Materials, Fujian, China) and were 2.7 mT for pulsed DC with a frequency of 50 Hz and 5.5 mT for the frequency of 100 Hz. In order to obtain uniform values of magnetic flux density, the two Helmholtz coils have been placed at a distance equal to half of their diameter (14.5 cm). The selection of the magnetic field frequency was based on the extensive review of the literature performed by Mattsson and Simko (2012) [2] which indicate that 50-100 Hz ELF-EMs have a perturbing effects on the cellular oxidative status, by both increasing the cellular reactive oxygen species and modulating the activity of catalase, peroxidase and glutathione peroxidase.

Figure 1

General schematics of the experimental set-up used for HRP (S) exposure to ELF-EMFs. A. The electronic scheme of the pulsed-DC generator. R1 and R2 are the approximate values of the Helmholtz coils resistance. If the switch S1-S2 is set to A position a pulsed-DC current with 100Hz frequency is obtained, while setting the switch to B a 50 Hz pulsed-DC current is obtained. B and C Oscilloscope view of a 50 Hz pulsed DC (B) and 100 Hz pulsed DC (C). The oscilloscope was set to 2 ms time base and 20 V/div.

2.3 Enzyme assay

HRP activity was measured by spectrophotometry using o-dianisidine as substrate following the indications from the Worthington Enzyme Manual [28]. Basically, a standard 1 ml reaction mixture consisted of various concentrations of H₂O₂, 0.8 mM o-dianisidine and 0.2 M phosphate buffer, pH 7. The reaction was started by adding 4 μg HRP and the mixture was exposed to ELF-EMS for 5 min. The enzymatic reaction was ceased by adding H₂SO₄ until approx. 30% was reached as a final concentration, and a pink colour was recorded at 540 nm using a DU730 Life Science Spectrophotometer (Beckman Coulter, Brea, CA, USA). The enzyme activity was expressed as μmoles oxidized o-dianisidine per min tacking into account an extinction coefficient for oxidized o-dianisidine of 1.13 x 10⁴ M^-1 cm^-1. Suitable controls for non-enzymatic oxidation of o-dianisidine as well as controls lacking H₂O₂ were performed and were found to be negligible (below 5% of the lowest recorded activity). The activity of unexposed HRP was monitored by maintaining the samples in the same condition as the exposed samples but with ELF-EMF system turned off. All measurements were performed at a temperature of 25 ± 0.4°C. To quantify the effect of ELF-EMF on the enzyme activity, the percent effect (PE, %) was calculated as using the equation described by Portaccio et al. 2005 [29]. Positive PE values indicate a positive effect of the EMF on the enzyme activity while negative PE values indicate a negative effect.

2.4 Kinetic parameters and statistical analysis

The Michaelis constant (K_m) and the maximum rate (V_max) were determined over a range of 5 μM to 1 mM H₂O₂ and the data were analysed using non-linear least-squares regressions using the DynaFit (BioKin, Ltd) software [30]. Regressions were fitted to Lineweaver–Burk plots. Additionally, the data were plotted using non-linear least-squares regressions to Eadie–Hofstee plots in order to confirm the results. Differences between the corresponding values generated from the three methods fall below 1%, in agreement with the report of Tseng and Hsu, 1990 [31]. The turnover number (k_cat) and the catalytic efficiency (k_cat/K_m) were calculated assuming a Mr of 44 kDa and a single catalytic centre per HRP molecule. All measurements were repeated five times and the data was analysed for statistical significance with one-way analysis of variance (ANOVA). Significant differences were determined by Tukey’s post hoc test [32].

3 Results

3.1 Effect of ELF-EMF on HRP activity

Two ELF-EMFs with different frequencies and intensities were applied on purified HRP and the effect on the catalytic process was recorded. As it can be seen in Figure 2, the application of a 100 Hz/5.5 mT ELF-EMF had no or little effect on the enzyme, the activity levels of the exposed HRP being similar with unexposed HRP at all substrate concentrations. Some slight modification in the activity could be observed, especially at high concentrations, but the differences were not statistically significant. Moreover, the average PE (%) was -0.22 ± 0.05 (%), too small to indicate any effect. The application of a 50 Hz/2.7 mT ELF-EMF on the other hand had a powerful inhibitory effect, with an average PE of -66.39 ± 1.08 (%). Not surprisingly, the same inhibitory effect although to a lesser extent was observed also by Portaccio et al. 2005 [29] after 4 h exposure of soluble HRP to a 50 Hz ELF-EMF. The same authors also indicate an activating effect of the 100 Hz ELF-EMF that our data failed to show.

Figure 2

Effects of ELF-EMF on HRP specific activity. The 50 Hz/5.5 mT ELF-EMF has a strong inhibitory effect on the HRP activity at all substrate concentrations. The experiment was repeated 5 times.

3.2 Effect of ELF-EMF on HRP kinetic parameters

One of the possible mechanisms by which the EMF can affect the enzymatic reaction is the induction of irreversible or slowly reversible modifications in the enzyme structure. These structural rearrangements would eventually alter the ability of the enzyme to bind or process its substrate(s). It has been shown that this is indeed the case for several enzymes when exposed for long times to EMFs, such as a cellulase from Trichoderma viride (exposed for 4 to 12 h to a static magnetic fields with various intensities [13]. To verify whether this is true also for the ELF-EMFs effect reported here, the HRP enzyme was incubated with various substrate concentrations while the magnetic field was applied. The data were fitted on the Lineweaver–Burk plot depicted in Figure 3 and the kinetic parameters V_max, K_m, k_cat and k_cat/K_m were calculated for both the unexposed and the exposed enzyme. Table 1 shows that there were no statistically significant differences in the K_m values between the control enzyme and the enzyme exposed to any of the two ELF-EMF used (P = 0.1), indicating that short exposure time did not managed to modify the affinity of HRP for H₂O₂.

Figure 3

Lineweaver-Burk plot depicting the influence of ELF-EMF on the K_m and Vmax of HRP. □ – unexposed HRP, ■ – HRP exposed to 100 Hz/5.5 mT ELF-EMF, ◊ - HRP exposed to 50 Hz/2.7 mT ELF-EMF. Each value represents the mean ± SE of five measurements.

Table 1

Comparison of the kinetic parameters for unexposed and ELF-EMF exposed HRP. The assay was performed at a o-dianisidine concentration of 0.8 mM and over a H2O2 concentration ranging from 5 μM to 1 mM. Values correspond to the average of five replicates ± standard deviation.

	Unexposed	100Hz / 5.5mT	50Hz / 2.7mT
Km (μM)	53 ± 11^a	67 ± 10^a	42 ± 11^a
Vmax (μmoles/min)	3.31 ± 0.17^a	2.75 ± 0.87^a,b	1.60 ± 0.19^b
kcat (s-1)	0.66 ± 0.03^a	0.58 ± 0.09^a,b	0.32 ± 0.04^b
kcat/Km (x103 s-1M-1)	13.48 ± 0.86^a	8.52 ± 0.42^b	7.15 ± 0.52^b

On the same line, mean values with different superscript letters indicate significant differences P < 0.05 as shown by the Tukey (HSD) analysis at a confidence range of 95.00 %.

A 50 Hz/2.7 mT EMF did however cause a significant decrease in the V_max value (3.31 ± 0.17 μmol/min vs. 1.60 ± 0.19 μmol/min, p < 0.03), which is also reflected in the turnover numbers (0.66 ± 0.03 s^-1 vs. 0.32 ± 0.04 s^-1, p < 0.05). The catalytic process performed by HRP is thereby affected by exposure to ELF-EMF, with the enzyme efficiency dropping to less than half when the catalytic process takes place under the 50 Hz/2.5 mT EMF. Although the same inhibitory effect, but to a lesser extent, was also seen for 100 Hz/5.5 mT ELF-EMF, the alterations of the V_max and k_cat values were not statistically significant (P = 0.09).

4 Discussion

A possible mechanism of ELF-EMs action reported in the literature is by directly generating free-radicals [27], which could serve as substrates for HRP or could catalyse the non-enzymatic oxidation of o-dianisidine. The fact that in our experiments both the non-enzymatic controls and the controls lacking H₂O₂ were negligible indicates that it is not the case, at least for ELF-EMFs exposure times of up to 5 min.

Nevertheless, similar alterations of the catalytic process induced by exposure to EMF have been observed for various other enzymes. Morelli et al. 2005 [33] reported that exposure to a 75 Hz EMF (with intensity varying from 240 μT to 2.5 mT) for up to 25 min did not modify the calculated K_m value for acetylcholinesterase, but dropped the V_max value to about 60%. The same authors also recorded an inhibitory effect of EMF exposure on alkaline phosphatase, phosphoglycerate kinase and adenylate kinase specific activity. A similar inhibitory effect on adenylate kinase upon application of a 75 Hz EMF with amplitudes above a threshold of about 125 μT have been reported also by Ravera et al. 2004 [15]. De Ninno et al. 2008 [11] showed that upon exposure to ELF-EMFs the specific activity of Cu²⁺, Zn²⁺ Superoxide Dismutase (SOD1) drops significantly, while the free radicals generation ability of a Fe²⁺ Xanthine Oxidase (XO) increases, both in manner related to the enzyme exposure-time and EMFfrequency. The reported ELF-EMFs ability to produce free radicals [27] and to modify the oxidative status of the cell could thereby be not a direct one, but indirect, by simply activating the ROS generating enzymes and decreasing the activity of the anti-oxidant enzymes.

All these enzymatic systems catalyse reactions in which a charged ion is involved, either as a charged substrate (quaternary ammonium ion of acetylcholine in the case of acetlycholinesterase), a metal ion implicated in catalysis (Zn²⁺ in alkaline phosphatase) or a metal ion associated with the substrate (Mg²⁺ in ATP in all the kinases). HRP makes no exception, as the catalytic mechanism involves a cycle between the Fe(III) resting state and some high oxidation state intermediates comprising an Fe(IV) oxoferryl centre and porphyrinbased cation radicals [24]. So the mechanism by which ELF-EMF influence the catalytic process must involve these charged ions. Indeed, since the 80`s it has been shown that when magnetic fields of given frequencies are applied to living organisms, variations in the ion concentrations within the cells can be recorded [34-35]. De Ninno et al. 2008 [11] postulated that the mechanism of coupling between ELF-EMF and living systems is based on a resonant frequency specific for a given ion molecule. At the resonant frequency, the signal is being picked up by the ions and could propagate to the whole enzyme structure influencing the catalytic mechanism by a yet unknown mechanism [11]. The resonant frequency for most metal ions can be calculated and it is in the range of 10–50 Hz, which is not that different from the frequencies reported so far which influence the enzyme’s activity.

Still, the methodology used to generate the ELF-EMs did not allow for the application of fields with the same intensity, but different frequencies. We are thereby hesitant to directly link the observed effects with only the frequency of the fields based solely on the reported data. An experimental set-up that would allow a better fine-tuning of ELF-EMF applied frequency (in unitary increments, or at least 5 Hz increments) and intensity is under development in our group.

5 Conclusions

The results presented in this study allow us to conclude that upon application of an ELF-EMF the catalytic efficiency of the Fe³⁺ containing HRP is diminished to less than half for a 50 Hz/2.7 mT EMF. These modifications of the kinetic parameters are not due to large structural alterations of the enzyme that might affect the substrate affinity, but most probably due to the interaction of EMFs with the charged ions involved in the catalytic process. The extent that these results have on the HRP in-vivo is debatable, as the conditions in which the ELF-EMFs were applied and the HRP activity was recorded are far enough from those occurring in a living cell. Still, one cannot ignore the fact that the modifications in the catalytic efficiency of the observed magnitude must perturb the economy of the cellular life and could be held responsible of the reported effects of ELF-EMFs on cellular oxidative status.

Acknowledgments

MM was supported by the GI-UAIC-02 awarded by the Alexandru I Cuza University of Iaşi.

Conflict of interest: Authors declare nothing to disclose.

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Received: 2015-8-19

Accepted: 2016-3-30

Published Online: 2016-5-6

Published in Print: 2016-1-1

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