Enzymes are essential for the catalysis of biochemical reactions and in the regulation of metabolic pathways. They function by greatly accelerating the rate of specific chemical reactions that would otherwise be slow. It has been shown that extremely low-power microwaves can influence enzyme activity [1¿5]. This study is focused at investigating the effects of low level microwave exposures ranging from 500MHz to 900MHz on L-Lactate Dehydrogenase (LDH) enzyme activity. The results obtained revealed the increased bioactivity of the LDH upon microwave radiation at two particular frequencies 500MHz and 900MHz

Cosic, I

Pirogova, E

Vojisavljevic, V

RMIT Research Repository

 
 
 
  
Abstract— Enzymes are essential for the catalysis of 
biochemical reactions and in the regulation of 
metabolic pathways. They function by greatly 
accelerating the rate of specific chemical reactions that 
would otherwise be slow. It has been shown that 
extremely low-power microwaves can influence enzyme 
activity [1-5]. This study is focused at investigating the 
effects of low level microwave exposures ranging from 
500MHz to 900MHz on L-Lactate Dehydrogenase 
(LDH) enzyme activity. The results obtained revealed 
the increased bioactivity of the LDH upon microwave 
radiation at two particular frequencies 500MHz and 
900MHz. 
 
INTRODUCTION 
The number of devices using microwave irradiation has 
been increasing the previous years rapidly, and the concern 
of military, industrial and government organizations with 
the possible health hazards associated with exposure to 
microwave irradiation was grown. The heating effect of the 
microwaves was already well-known but a doubt remained 
on the existence of a non-thermal effect. Isolated enzymes 
in aqueous medium were taken as model to determine if 
there was an effect of the radiation on the biological 
processes. These first studies have been carried out with 
various microwave apparatuses, with frequencies and 
powers of irradiation used varied significantly from one 
study to another [1].  
Thirty years ago, Frohlich postulated that extremely 
weak microwave signals may act as a trigger for certain 
bioeffects. During the last few decades, the use of 
microwave radiation has greatly increased in radar and 
communication systems as well as in food-processing 
technology and in other industrial applications. The 
development of consumer and medical microwave devices 
for clinical diagnosis and therapy also has prompted 
widespread interest and has stimulated much research on 
the mechanisms of interaction of microwave radiation with 
living organisms [2-5]. 
 
Manuscript received April 7, 2008. This work was proudly supported 
by the Australian Centre of Radiofrequency Bioeffects Research 
(ACRBR).  
E. Pirogova is with the School of Electrical and Computer 
Engineering, RMIT University, GPO Box 2476V Melbourne Victoria 
3001 Australia (corresponding author: tel. 61-3-9925-3015, fax 61-3-
9925-2007, elena.pirogova@rmit.edu.au) 
V. Vojisavljevic is with the IM Medical Ltd, 484 St Kilda Road 
Melbourne VIC 3004 Australia (vvojisavljevic@immedical.com.au).  
I. Cosic is with the School of Electrical and Computer Engineering, 
RMIT University, GPO Box 2476V Melbourne Victoria 3001, Australia 
(e-mail: irena.cosic@rmit.edu.au). 
According to the literature, two types of effects can be 
ascribed to microwaves, i.e. thermal and non-thermal [2-5]. 
The thermal effects are related to the heat generated by the 
absorption of microwave energy by the water medium or 
by organic complex systems, both characterized by a 
permanent or induced polarization. At present, very little is 
known about the molecular mechanisms involved in the 
putative non-thermal effects which could involve direct 
energy transfer from the electromagnetic field to the 
vibrational modes of macromolecules altering their 
conformation [6-7]. 
Biological molecules, once perceived as rigid structures, 
are now known to show rapid, continuous changes in shape 
that may well be important in their biological functions. It 
was reported that microwaves radiation may be used to 
selectively manipulate biochemical processes remotely and 
non-thermally [8]. Therefore, proteins, and more 
specifically enzymes, present themselves as target systems 
for microwave electromagnetic radiation. Experiments 
have shown that extremely low-power microwaves can 
affect enzymes activity. For example, Bose reports a 
tripling in the rate at which the enzyme cellulose breaks 
down cellulose compounds with the enhancing effect of 
microwave radiation [9]. Also reported is the accelerated 
hydrolysis of albumin [10], and the rapid disruption of a 
peptide bond cleavage [11]; neither of which can be 
attributed to thermal heating effects. It was suggested that 
two particular parameters seem to influence the enzymatic 
activity under microwave irradiation: the hydration state 
and the polarity of the reaction medium. 
However, the results obtained on enzyme systems are so 
far controversial, probably due to the experimental 
difficulties in the proper control and monitoring of the 
temperature. No measurable non-thermal effect on catalytic 
activity has been observed in a number of isolated enzymes 
irradiated in vitro [9-12]. Conversely, other enzyme 
systems such as lymphocyte protein kinases [13], hepatoma 
cell ornithine decarboxylase [14] and acid phosphatase [15] 
respond to low or high intensity and amplitude-modulated 
microwave fields. Moreover, a significant inhibition of red 
cell Na/K. ATPase, presumably related to conformational 
changes of the protein, has been reported [16]. 
In our previous studies [17,18] we have tested the 
influence of visible light in a range of 550-850 nm and 
infrared radiation ranging from 1140 up to 1200 nm on 
enzyme kinetics of the LDH enzyme. This study extends 
our investigation of non-thermal biological effects of low 
power electromagnetic radiation (EMR) exposures on the 
LDH biological activity in the microwave range. In 
particular, this paper reports on results of our experimental 
Non-thermal Effects of 500MHz – 900MHz Microwave Radiation on 
Enzyme Kinetics 
 
E. Pirogova, V. Vojisavljevic, and I. Cosic, Senior Member, IEEE 
30th Annual International IEEE EMBS Conference
Vancouver, British Columbia, Canada, August 20-24, 2008
978-1-4244-1815-2/08/$25.00 ©2008 IEEE. 1052
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study of the LDH enzyme aqueous solutions exposed to 
500MHz – 900MHz microwave radiation with the power 
of -50dBm, -40dBm and -30dBm (0.01µW, 0.1µW and 
1µW respectively). 
I. MATERIALS AND METHODS 
A. Microwave exposure system 
As a source of microwave radiation we used TC-5062A 
UHF TEM Cell (100kHz – 3GHz), TESCOM Ltd (Pic. 1). 
This TEM Cell generates the electromagnetic field for 
irradiating the object of investigation. Through the input 
port, an external signal is applied to generate predictable 
field inside the TEM Cell.  
 
 
Picture1. Sample exposure system (TC-5062A UHF TEM Cell) 
 
A GTEM (model TC-5062A) was used to expose the 
enzyme solution samples.  The GTEM was calibrated using 
a broadband electric field probe to determine the electric 
field produced at the sample position inside the GTEM for 
a given input power.  For the input powers used in this 
experiment the test fields at each frequency are shown in 
Table 1. 
 
We scale the field values we provide for 10 dBm (10 mW) 
input to the power levels we used, using: 
 
E1 = E0*sqrt(P1/10mW) 
 
Where E1 is the exposure field of the sample (that we 
would like to know), E0 is the calibration field we found 
using a test power of 10 mW, and P1 is the test power we 
used in our exposure.  The results have shown that the 
estimated uncertainty in the test field is ± 30%. 
 
B. Experimental study 
Dehydrogenases are the enzymes that catalyze a variety 
of oxidation-reduction reactions within the cells. As the 
protein example we have chosen the L-Lactate 
Dehydrogenase (rabbit muscle, EC1.1.1.27). This enzyme 
has been selected fort this experimental investigation on 
the basis of its commercial availability, simplicity of the 
assay, and the possibility of measuring its bioactivity using 
the standard, well accepted procedure, i.e. Continuous 
Spectrophotometric Rate Determination. To measure the 
absorption coefficients of the analyzed samples we have 
used an Ocean Optics USB2000 spectrometer coupled with 
a CCD array, which can detect in the 190-870 nm range 
(Pic. 2). Software control automatically monitors and saves 
the optical density values (absorption coefficients) at the 
required wavelength every 30 sec.  
 
 
Picture 2. Spectrophotometer USB2000 
 
L-lactate dehydrogenase (LDH) catalyses the inter-
conversion of the l-lactate into pyruvate with the 
Nicotinamide Adenine Dinucleotide Oxidised 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 340 nm in contradiction to the NAD, which is inactive at 
this frequency. Due to the different optical characteristics 
of the NADH and NAD we are able to optically asses if the 
reaction of conversion Pyruvate → Lactate in the presence 
of the LDH as an accelerator has occurred and then 
determine the amount of the reactants.  
 
Assay preparation: 
2.5 ml cuvettes are filled with the following 
components:  
- 0.1 ml of 0.0027M Sodium Pyruvate (BioWhittaker);  
- 0.1 ml NADH; disodium salt (C21H27N7O14P2Na2 
Roche);  
- 0.005M Phosphate buffered saline (SIGMA);  
- 1.5 ml of deionized water;  
- 0.3ml LDH diluted in 2mg/ml of Phosphate buffered 
saline with BSA (SIGMA). 
 
The experimental procedure: 
1. The cuvettes were filled with 0.3ml of the LDH 
samples. The samples were irradiated for 5 min using the 
GTEM exposure system set at the activation microwave 
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frequencies ranging from 500 MHz to 900 MHz and 
radiation power of -30dBm, -40dBm and -50dBm (1µW, 
0.1µW and 0.01µW respectively). 
2. The irradiated samples were then added to the already 
prepared solution of NADH and pyruvate. We have diluted 
the stock coenzyme solution with the 0.003 M potassium 
phosphate assay buffer.  
3. The spectrophotometer was set to 100% transmittance 
(zero absorbance) at each wavelength using the 0.003 M 
K2HPO4 assay buffer blank.  
4. Using the spectrophotometer, the absorption 
coefficients of NADH (rate of change in absorbance) were 
measured at 340 nm. The measurements were repeated 
three times for each analyzed radiating frequency and 
power values. The obtained data were collected and 
presented in Table II and Fig. 1.  The experiments were 
performed at room temperature 27°C (Temperature 
controller, Quantum Northwest).  
 
II. RESULTS AND DISCUSSION 
The results obtained have shown the change in NADH 
absorbance of analyzed samples under the influence of the 
irradiated LDH enzyme. From Fig.1 we can observe that 
after being irradiated by microwaves at the specific 
frequencies 500MHz and 900MHz, the LDH bioactivity 
has increased resulting in accelerating the reaction: 
 
         LDH 
      Pyruvate + NADH ------> Lactate + NAD+ + H+.  
 
0.000
0.004
0.008
0.012
0.016
0.020
no
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irr
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50
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-4
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0/
-5
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-4
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-5
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-5
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F re quenc y [MHz ]/P ower [dB m]
ra
te
 o
f 
ch
an
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e
 
Figure 1. Rate of change in NADH absorbance upon microwave 
irradiation of the LDH enzyme 
 
The results obtained revealed (Fig.1, Table II) that the 
maximum value of NADH optical density/rate of change in 
NADH absorbance is 0.0157 (achieved at the frequency 
900MHz and power of -30dBm), which corresponds to 
73% increase in comparison with the rate of change value 
0.0091 of the non-irradiated enzyme. Moreover, from 
Table II and Fig.1 we can also observe that microwaves at 
500MHz/-30dBm, 500MHz/-40dBm and 500MHz/-50dBm 
have also affected the LDH bioactivity that resulted in the 
increased rate of change in NDAH absorbance. These 
values (0.0132, 0.0133, and 0.0131 respectively) are higher 
(45%, 47%, and 43%) than the rate of change 0.0091 in the 
presence of the non-irradiated LDH (Table II).  
Interesting to note, when the LDH enzyme was 
irradiated by all other selected microwave frequencies 
650MHz, 700MHz and 750MHz (Table II, Fig.1), the 
measured  NADH absorption coefficients were lower than 
in the case of non-irradiated enzyme. Thus, we can 
conclude that the microwave exposures at these specific 
frequencies 650MHz, 700MHz and 750MHz have also 
affected the LDH kinetics leading to the decreased rate of 
change in NADH absorbance (2%-15% depending on the 
irradiating frequency), i.e. have slow down the overall 
chemical reaction (Table II). 
III. CONCLUSION 
In our previous work it was shown that enzyme activity 
can be modulated by electromagnetic radiation at the 
specific frequency of visible and infrared light. Here we 
have shown that the biological activity of the studied LDH 
enzyme can be increased by 40%-70% using low power 
microwaves at 500MHz/-30dBm, 500MHz/-40dBm, 
500MHz/-50dBm and with the maximum increase 
achieved at 900MHz/-30dBm.  
On the other hand, we have observed the opposite effect 
with the microwave frequencies of 650MHz, 700MHz and 
750MHz (radiation power of -30dBm, -40dBm and -
50dBm), when the LDH activity was decreased by 2%-
15%.  
Hence, the results suggest that this specific biological 
process can be modulated by the defined frequencies of 
microwave radiation. The possibility of influencing on 
protein biological activity using microwave radiation 
would benefit the development of new biomaterials, non-
invasive treatments and advanced technologies.  This could 
have major implications in drug design, medicine, 
agriculture, pharmacology and biotechnology.  
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of microwave radiation on enzymatic properties: application in 
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[2] M. Porcelli, G. Cacciapuoti, S. Fusco, R. Massa, G. D’Ambrosio, C. 
Bertoldo, M. De Rosa, V. Zappia, (1997) “Non-thermal effects of 
microwaves on proteins: thermophylic enzymes as model system”, 
FEBS Letters, 402, 102-106. 
[3]    H. Frohlich, (1980) Adv. Electr. Electr. Phys. 53, 85-152. 
[4]    E. Marani, H.K.P. Feirabend, (1994) Eur. J. Morphol. 32, 330-334. 
[5]    J.L. Kirschvink, (1996) Bioelectromagnetics, 17, 187-194. 
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[7]  E.E. Fesenko, V.I. Geletyuk, V.N. Kazachenko, N.K. Chemeris, 
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[8] B. Ellison, C. Gibson, N.A. Grant, G.T. Hylord, J.T. Magee, D. 
Pooley, B. Stewart, (1999) „An investigation into application of 
microwave radiation“, Proc. SBMO/IEEE MTT-SIMOC’99, 57-62. 
[9]  G. Whittaker, (1998) “Microwave effects”, New Scientist, 34-37. 
[10] A. Morales-Rubio, (1993) “Rapid acid hydrolysis of albumin in a 
microwave oven”, Microchemical Journal, 47, 270-277. 
[11] Chi-Yue Wu, (1992) “Specific peptide-bond cleavage by 
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[12] R. Spencer, (1985) Journal of Medical Microbiology, 19, 269-272. 
[13] M. Dreyfuss, J. Chipley, (1980) Applied and Environmental 
Microbiology, 39, 13-16. 
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[14] S. Webb, D. Dodds, (1968) “Inhibition of bacterial cell growth by 
136GHz microwaves”, Nature, 218, 374-376. 
[15] W. Grundler, F. Keilman, (1983) “Sharp resonances in yeast growth 
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[16] A. Sevast’yanova, (1974) “A study of the effects of millimeter 
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[17] V. Vojisavljevic, E. Pirogova, I. Cosic, (2007) “The effect of 
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[18] V. Vojisavljevic, E. Pirogova, I. Cosic, (2007) IEEE EMBS  
 
 
 
 
TABLE I. GTEM exposure system calibration results 
Freq (MHz) P (dBm) E (means) P (mW) E (10dBm) d (m) 
500 9.53 2.01 8.974288 2.121759 0.333264 
600 9.48 2.24 8.871560 2.378198 0.297329 
700 9.45 2.53 8.810489 2.695383 0.262340 
750 9.46 2.73 8.830799 2.905110 0.243401 
800 9.48 3.29 8.871560 3.492978 0.202437 
9.46 9.46 4.21 8.830799 4.480042 0.157835 
 
 
 
TABLE II. Frequency and power values of microwave radiation, and rate of change in absorbance 
Frequency, MHz Power, dBm 
Rate of change in 
NADH absorbance 
Relative 
difference 
    
 Averaged values and 
(max-min) 
irrad. vs non-
irrad. (%) 
non-irradiated 0 0.00911 0% 
    
500 -30 0.0132 (0.0007) 45 
500 -40 0.0133 (0.0010) 47 
500 -50 0.0131 (0.0002) 43 
    
650 -30 0.0077 (0.0012) -15 
650 -40 0.0085 (0.0003) -6 
650 -50 0.0081 (0.0002) -10 
    
700 -30 0.0083 (0.0020) -9 
700 -40 0.0086 (0.0011) -6 
700 -50 0.0087 (0.0004) -4 
    
750 -30 0.0081 (0.0002) -11 
750 -40 0.0076 (0.0016) -16 
750 -50 0.0082 (0.0003) -10 
    
900 -30 0.0157 (0.0027) 73 
900 -40 0.0089 (0.0011) -2 
900 -50 0.0089 (0.0002) -3 
 
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Non-thermal effects of 500 MHz-900MHz microwave radiation on enzyme kinetics

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