Review of Studies on Modulating Enzyme Activity by

32nd Annual International Conference of the IEEE EMBS Buenos Aires, Argentina, August 31 - September 4, 2010

Review of Studies on Modulating Enzyme Activity by Low Intensity Electromagnetic Radiation Vuk Vojisavljevic, Elena Pirogova, and Irena Cosic, Senior Member, IEEE

Abstract—This paper is a compilation of our findings on nonthermal effects of electromagnetic radiation (EMR) at the molecular level. The outcomes of our studies revealed that that enzymes’ activity can be modulated by external electromagnetic fields (EMFs) of selected frequencies. Here, we discuss the possibility of modulating protein activity using visible and infrared light based on the concepts of protein activation outlined in the resonant recognition model (RRM), and by low intensity microwaves. The theoretical basis behind the RRM model expounds a potential interaction mechanism between electromagnetic radiation and proteins as well as protein– protein interactions. Possibility of modulating protein activity by external EMR is experimentally validated by irradiation of the L-lactate Dehydrogenase enzyme.

I. INTRODUCTION

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HE electromagnetic (EM) force is one of the four fundamental forces in the universe. Technological progress is associated with a growing number of artificial electromagnetic fields (EMFs) in the environment and these EMFs can interfere with basic biological processes affecting the health of the population. Hence, research into the interactions of artificial EMFs with biological systems and possible health consequences has become a priority area for national and international health organizations. Non-ionizing artificial electromagnetic radiation (EMR) from extremely low frequencies (50/60 Hz) to visible light (1014-1015 Hz) is increasing in the environment. Our current understanding of how EMR of different frequencies affects biological systems needs to be considerably enhanced. For example, electrical activity of the brain and heart may be affected by low frequency EMR, while higher frequencies may affect cells, ion channels and biomolecules. It has been shown that light-activated changes in protein energy states can induce or modulate biological processes. For instance, light-activated excitation of rhodopsin (bacteriorhodopsin) molecules, involved in the Manuscript received April 1, 2010. This work was supported by the RMIT Health Innovations Research Institute. V. Vojisavljevic is with the School of Electrical and Computer Engineering, RMIT University, GPO Box 2476 Melbourne 3001 Victoria Australia, phone: 61-3-9925-3077; fax: 61-3-9925-2007; e-mail: [email protected]. E. Pirogova is with the School of Electrical and Computer Engineering and Health Innovations Research Institute, RMIT University, GPO Box 2476 Melbourne 3001 Victoria Australia, phone: 61-3-9925-3015; fax: 613-9925-2007; e-mail: [email protected]. I. Cosic is with the Science, Engineering and Health College and Health Innovation Research Institute, RMIT University, GPO Box 2476 Melbourne 3001 Victoria Australia, phone: 61-3-9925-9903; e-mail: [email protected]

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hyperpolarization process of a cell membrane, can either generate nerve impulses, ATP synthesis, or regulate embryogenesis [1–5]. It has been also suggested that cytochrome c oxidase and certain dehydrogenases may play a key role in the photoreception process, particularly in the near infra-red (NIR) frequency range [5]. Recent studies into effects of low-intensity non-thermal light irradiation (pulsed and continuous) on eukaryotic and prokaryotic cells, reported the accelerated proliferation rate in yeast and mammalian cells upon irradiation by He-Ne laser light [6,7] and increased E. coli proliferation rate by argon laser light exposures [5,7], and by laser light exposures of 1–50 J/cm2 at wavelengths of 630 and 810 nm [8] on various bacteria. Several studies reported changes in the activity of human erythrocytes after low intensity light radiation at 810 nm. Our research was aimed to study whether EMFs of different frequencies and intensities can modify protein (enzyme) kinetics. We investigated the influence of visible light in the range of 550nm-850nm and infrared radiation in the range of 1140nm-1200 nm on LDH enzyme kinetics [9,10]. The experimental evaluation of EMR effects on LDH enzyme activity was based on computational predictions using the Resonant Recognition Model (RRM) [1,11]. By utilizing the RRM we can identify a particular frequency in the range of visible (VIS) and NIR light that can lead to protein activity modulation [1,9]. The number of devices using microwave irradiation has been increasing rapidly in recent years, and the concern of military, industrial and government organizations with the possible health hazards associated with exposure to microwave irradiation has grown. Isolated enzymes in aqueous medium were taken as models to determine if there is an effect of low intensity microwaves on biological processes. Thus, in addition we studied non-thermal bioeffects of microwaves in the range of 500MHz to 900MHz on LDH biological activity. II. MATERIALS AND METHODS A. Resonant Recognition model (RRM) The RRM was designed for the analysis of protein (DNA) interactions and their interaction with EMR [1,11]. The RRM model can analyze protein primary structures, i.e. amino acid sequences, using digital signal analysis methods that include spectral and space-frequency analyses. It has been found that the distribution of the energies of free electrons along a protein molecule is critical for the protein

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function (interaction) [1,11].The RRM theory states that an external EMF at a particular frequency would produce resonant effects on a protein’s biological activity [1,9,11]. It has been shown that all protein sequences with a common biological function have a common frequency component in the free energy distribution of electrons along the protein backbone. This characteristic frequency is related to the protein biological function [1,9]. According to the RRM, protein interactions can be viewed as a resonant energy transfer between the interacting molecules. This energy can be transferred through oscillations of a physical field possibly electromagnetic in nature [1,11,12]. Since there is evidence that proteins have certain conducting or semiconducting properties, a charge, moving through the protein backbone and passing different energy stages caused by different amino acid side groups, can produce sufficient conditions for specific electromagnetic emission or absorption [1,12]. The frequency range predicted for protein interactions is from 1013 Hz to 1015 Hz. This estimated range includes infrared (IR), visible and ultraviolet (UV) light. A linear correlation between the peaks in absorption spectra of proteins and their RRM characteristic frequencies has been established. The computationally identified characteristic frequency for a protein functional group can be used to calculate the wavelength of applied irradiation, λ, which assumingly would activate this protein sequence and modify its bioactivity λ =K/fRRM (K=201 nm, K is a scaling factor) [1,9]. In our study the RRM was used for structure-function analysis of 32 Dehydrogenase enzyme sequences. A multiple cross-spectral analysis was performed resulting in two characteristic frequencies identified at f1=0.1680±0.004 and f2=0.2392±0.004 (Fig.1), which correspond for the biological activity of the analyzed dehydrogenase protein group. We then calculated the wavelength of irradiation (λ=201/fRRM), which would modulate dehydrogenase enzyme bioactivity: λ1=1156±15nm and λ2=846±15nm. In the experimental study the LDH samples were irradiated by the VIS (550nm - 850nm) and NIR (1140nm-1200nm) light, and LDH activity was measured before and after the exposures [9,10]. B. Irradiation of L-Lactate Dehydrogenase enzyme using VIS and IR light Enzymes are proteins crucial in accelerating metabolic reactions in living organisms. Dehydrogenase enzymes catalyse a variety of oxidation-reduction reactions within cells. As the protein example we have chosen L-lactate dehydrogenase (rabbit muscle) due to its well known characterization, and the possibility of measuring its activity using the standard procedure, the so called. Continuous Spectrophotometric Rate Determination. This methodology was employed in this study due to a possibility of monitoring the “reaction rate” changes in real time. LDH (rabbit muscle) catalyses the inter-conversion of the L-lactate into pyruvate with the nicotinamide adenine

dinucleotide oxidized form (NAD+) acting as a coenzyme. The suitability of the LDH enzyme for this reaction is attributed to the absorption characteristics of the NADH (nicotinamide adenine dinucleotide reduced form). NADH is able to absorb light at 340nm contrary to the NAD, which is inactive at this frequency. f1

f2

Fig. 1. Multiple cross-spectral function of Dehydrogenase proteins (32 sequences). The prominent peak(s) denote common frequency components. The abscissa represents the RRM frequencies and the ordinate is the normalized signal intensity [9].

Due to different optical characteristics of NADH and NAD we are able to optically assess if the reaction pyruvate → lactate in the presence of the LDH as an accelerator has occurred and then determine the amount of the reactants [9]. As a source of IR and visible light we have used a SpectraPro 2300i monochromator (Acton Ltd) with a wavelength range of 400nm–1200nm and a resolution of 0.1 nm. For measurement of absorbance of the analyzed enzyme solutions we used an Ocean Optics USB2000 spectrometer, with the range of 190 nm–870 nm. C. Effects of low intensity microwaves on enzyme activity We also studied [13] the effects of low level microwaves on LDH enzymatic activity. The activity of LDH was calculated from absorption curves using the “gradient methods”. The enzyme solutions were exposed to low intensity microwave radiation using a TC-5062A TEM Cell, TESCOM Ltd. The frequencies of applied radiation were in the range 400MHz - 975MHz and enzyme activity was monitored (by measuring optical density of the LDH solution) at steps of 25 MHz. Measurements were repeated 3-8 times depending on their standard deviations, irradiating frequency and electrical field values. Enzyme activity measurement The assay contained the following components: 3.00 ml reaction mix includes 10 mM sodium phosphate, (SIGMA, USA), 0.12 mM–NADH, disodium salt (ROCHE, Germany) reduced form, 2.3 mM pyruvate (BioWhittakerTM, USA), 0.033% (w/v) of bovine serum albumin (SIGMA) and 0.05 units of L-lactic dehydrogenase (ROCHE). The LDH kinetics was measured by monitoring of NADH absorption at 340nm. The activity of the enzyme examples is determined by the rate of substrate utilization during the enzyme catalyzed reaction.

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B. Effects of near infra-red radiation on enzyme activity The experiments were performed at room temperature 27°C (Temperature controller, Quantum Northwest). The cuvettes were filled with 0.3ml of the LDH samples. The samples were irradiated with NIR light of different wavelengths (1140nm-1200nm) for 600 sec. These irradiated samples were added then to the already prepared solutions of NADH and pyruvate.

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Fig. 2 Summary of the effects of EMR on LDH activity. A bar graph +/- SD for no irradiation, irradiation at 586 nm, 829 nm and 650 nm shows that EMR of visible and IR light does modulate LDH activity. No effect was observed for irradiation between 575 and 580 nm; 600 and 645 nm; 665 and 810 nm. Y axis corresponds to ‘Relative LDH activity’ with 100% corresponding to the activity in the absence of irradiation. 1.600 114 0

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A. Effects of VIS light on enzyme activity We investigated the effects of visible light in the range of 550nm - 850nm and infra-red in the range of 1140nm 1200nm on LDH kinetics [9,10]. Data was collected and presented in Figure 2. The effects of light exposures on LDH activity are measured as the rate of change of the NADH concentration per second. The enzyme solutions were irradiated for 15 min and LDH enzyme activity was measured immediately after irradiation. From Fig. 2 an evident increase in the LDH activity after irradiation by visible light at the particular wavelengths of 829 nm and 595 nm (Fig. 2) can be observed. However, there is no significant difference in activities of LDH samples irradiated by light of other wavelengths and the control (non-radiated) solutions. In comparison to the nonradiated LDH solutions that have average rate of 0.022 with a standard deviation of ± 0.0015, the results obtained demonstrate the increase of LDH activity in order of 11.9% (P < 0.001) at 596 nm and 12.67% (P < 0.001) at 829 nm respectively [9]. To evaluate how significant is the difference between the mean values of the activity of irradiated and non-irradiated samples, we have used an independent two-sided T-test. The results obtained reveal that the computationally predicted activation frequencies of LDH enzymes using the RRM approach closely correspond to our experimental data and findings of other researchers, showing that the maximum change in LDH activity upon irradiation occurs at 596 nm and 829 nm (Fig.2). Hence, the results obtained reveal that this specific biological process can be modulated by irradiation with the defined frequencies, thus strongly supporting the main concept of the RRM methodology.

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III. RESULTS AND DISCUSSIONS

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Optical activityat 340nm

The temperature of experimental solution was controlled during the enzyme sample irradiation and activity measurement procedures. Enzyme concentration was determined by the extinction coefficient. For each irradiated sample we measured the absorption spectra for 10 min by recording the solution’s absorption values every 30 sec. For each wavelength of light radiation we undertook 3-5 control experiments to measure the activity of non-radiated protein solutions. The results obtained were analyzed and the enzymatic activity of non-radiated solutions was compared to the activity of the irradiated samples (calculation of the P value) [9,10].

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Fig. 3 Changes of absorption coefficient values (at 340 nm) in time after irradiation with different wavelengths [10]

The optical density of NADH was measured at 340nm for each irradiating wavelength. The results obtained revealed the change of NADH absorbance under the influence of irradiated LDH [10]. From Fig.3, we can observe that maximum optical density of the NADH is achieved at the wavelengths 1192nm and 1200nm (f1=0.1688±0.004) as was predicted by the RRM as the possible activation frequency of the dehydrogenase enzymes. Hence, the results suggest that this specific biological process can be modulated by irradiation with defined frequencies. C. Effects of low intensity microwaves on enzyme activity The absorption coefficients of the studied enzyme samples were measured by an Ocean Optics USB2000 spectrometer. Sampling rate for the absorption coefficient at 340 nm was set at 1 sec. The experimental solutions were prepared according to the assay provided by Sigma-Aldrich. The enzyme solutions were exposed (sham exposed) to microwave radiation, which was generated by the certified TEM-cell with a signal generator (ROHDE&SCHWARZ SMX). The EMF inside the test volume is proportional to the input voltage and inversely proportional to the cell height. Although, we cannot distinguish which field is more responsible for non–thermal effects, we suggest that one of the main effects, i.e. the change in a protein’s hydration, can be induced by an applied EMF [13]. The cuvettes were filled with the sample and kept at 22 cm distance from the top of the camera. The exposure duration was 240 sec. After exposure, the optical density of the NADH was recorded continuously for the next 5 minutes with the step of 1 sec. The control cuvettes were also kept

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under the same experimental conditions inside the camera with the signal generator being in the ‘switched-off’ mode (sham exposed). The frequencies of the applied microwaves were in the range of 400 MHz - 1800 MHz with a step of 25 MHz.

IV. CONCLUSION In study non-thermal effects of VIS (550nm–850nm) and NIR (1140nm-1200nm) on LDH enzyme kinetics were investigated. In addition, we also irradiated the LDH samples using low intensity microwaves (400MHz - 975MHz). The results showed that applied low intensity EMR can induce some frequency specific alterations in conformation of the studied LDH enzyme. These observed biological effects can be explained by possible resonant effects produced by external EMR at the molecular level and thus, could lead to some health effects and not necessarily harmful but therapeutic. ACKNOWLEDGMENT

Fig. 4. Rate of change in NADH absorbance upon microwave irradiation of the LDH enzyme [13]

For each frequency of irradiation the effects were measured for the following electrical fields: 0.02135 V/m, 0.06754 V/m, 0.21358 V/m, 0.675 V/m and 2.136 V/m (0.0000012 W/m2, 0.000012 W/m2, 0.00012 W/m2, 0.0012 W/m2, and 0.012 W/m2 respectively). The data obtained was collected; and the significance of the data was assessed using a single factor ANOVA analysis. For each frequency and power we tested H0 hypothesis that variability among the irradiated and non-irradiated samples represents a random error. For the hypothesis being tested we set α=0.05. The results showed [14] that biological activity of the LDH enzyme increased by 5%-10% using low intensity microwaves at 500-525 MHz for electric fields of 0.021-2.14 V/m. We also showed that 900MHz irradiation increased LDH bioactivity for electric fields in the range of 0.021 V/m0.068 V/m and decreased activity if the field was stronger than 0.67 V/m. We observed a slight inhibiting effect on LDH activity at the frequencies of 650 MHz (0.214 V/m); 700 MHz (0.68-2.14 V/m) and for 875-925 MHz (0.68-2.14 V/m), when LDH activity was decreased by 2%-15%. The experiment was specifically designed to measure the effects (changes in absorption coefficients of solutions) induced by microwave irradiation of different frequencies and intensities. During the experimental procedure the temperatures of the experimental solutions were monitored. We observed only a change in the temperature of the solution less then 0.01 K. Due to small intensity of microwaves used in this study, there is no heating observed, and no differences among the temperatures of the analyzed samples were recorded. In summary, we observed the maximum increase in LDH activity at two particular frequencies 500 MHz and 900 MHz (Fig. 4). The outcomes of this study show that low intensity microwaves of particular frequencies can induce changes in enzyme bioactivity and thus, affect the specific biological process involving the LDH enzyme.

This study was funded by the Australian Centre for Radiofrequency Bioeffects Research and the RMIT Health Innovation Research Institute. REFERENCES [1] [2] [3]

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