In: Mobile Telephones... ISBN: 978-1-60456-436-5 Editors: A.C. Harper and R.V. Buress, pp. 11-26 © 2008 Nova Science Publishers, Inc. ALTERATION OF ENZYMIC ELECTRON TRANSFER REACTIONS INDUCED BY MICROWAVES EMITTED BY GSM MOBILE PHONE Roberta De Carolis', Fiorenzo Marinelli’ and Mario Barteri’” ‘Department of Chemistry, University of Rome “La Sapienza”, Italy “Inst. of Molecular Genetics, Bologna, Italy Abstract Enzymatic activity and conformational features of bovine lactoperoxidase (LPO), ascorbate oxidase (AO) and laccase (LAC) are modified when aqueous solutions of these enzymes are exposed to the microwave field emitted by a mobile phone. Several kinetic and spectrophotometric measurements were carried out comparing native and irradiated samples of the same enzyme solution. Experimental data obtained demonstrate that mobile phone radiations alter the active site conformational state. It has been also proved that electromagnetic fields (EMFs) interfere with free radical intermediate species of the electron transfer enzymatic reaction, providing a considerable modification of the kinetic parameter Kur, Vas and Kear Introduction The global diffusion of mobile communications is enormously increased into our everyday life, such that it is estimated that, nowadays, there are more than 1.5 billions mobile phones in use. Due to the microwave character of mobile telephone emissions, it was suggested that phone emissions might affect biological systems. A number of studies have attempted to determine if (and how) exposure to microwaves (MWs), under conditions similar to GSM emissions, can induce alterations in the kinetic of the folding process of globular proteins [1-2]. In our laboratory it has been shown as MWs significantly modify kinetic and structure of acetylcholinesterase (AChE), an important enzyme which regulates the activity * Author to whom correspondence should be addressed.12 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri of the neurotransmitter acetylcholine in the brain [3]. The conceptual starting point of these our previous studies foresaw the use of highly purified bio-macromolecules (proteins, enzymes and nucleic acids) as molecular probes, to check possible kinetic and/or conformational effects induced by MWs exposure. We supposed that any possible biological damages could start at “molecular level,” involving biological macromolecules. We believe conceivably true that the in vivo effect of conformational and/or kinetic alteration in proteins and enzymes may be progressively propagated to higher and to more complex levels of the biological systems, modifying biochemical reactive pathways or membrane activities. Many prestigious laboratories have been interested in. studying biological effects induced by exposure to MWs in the working range of mobile phone. These studies have often obtained conflicting results leading to contrasting conclusions. Furthermore, the use of commercial mobile phones as a MWs source has been criticized, since phone emissions vary widely [4]. This variability is generally considered as a huge drawback originating irreproducible experimental data and providing incongruent results. However, several studies carried out using certified emission sources (TEM chambers and/or antennas) on biological systems, such as specific cell strain, selected tissues and genetically modified mice, also yield irreproducible effects. The main reason of this incongruence should be probably searched in the intrinsic complexity of biological systems. Even a single cell is a very complicated system made up of a precise and synergic assembly of phospholipids, nucleic acids, proteins and enzymes. The specific and peculiar distribution of these compounds rules all the cellular biological activity, including the ability of repairing or masking possible damages. Thus, the topological distribution of cellular biomolecules, with different chemical composition and different conformational states, are important in the specific interaction with electromagnetic fields (EMFs). For instance, the electrostatic interactions of EMFs with phospholipid-bilayers of cellular membrane are different if compared to those with a protein. On these macromolecules the charge distribution can cause conformational and functional modification of the biological activity. Our research strategy consisted in using aqueous solutions of specific and highly purified metallo enzymes and proteins to study the EMFs effects at a molecular level. We selected proteins that fitted at least one of the following criterions: proteins with a high dipole momentum ( 500 Debye), metallo-enzymes containing at least one paramagnetic metal ion, or enzymes which catalyzes electron-transfer reactions with a mechanism involving paramagnetic intermediate species, such as free radicals. In this chapter, we report some results concerning the effects of mobile phone microwaves on structure and activity of metallo-enzymes. Our studies were devoted to demonstrate that the mobile phone emission interferes with electron transfer processes, which take place during enzymic reactions catalyzed by Oxidases and Peroxidases. Biochemical reactions catalyzed by these enzymes proceed generating free radicals intermediate compounds [5], which are paramagnetic species sensitive to electromagnetic fields. Here we show experimental evidences proving that the microwaves emitted by a dual band mobile phone (915-1822 MHz) alter both conformational and configurational features of steady-state transition complex of the enzymatic reaction mechanism. Figure 1 shows the electric field emissions of the mobile phone recorded operating in both receiving and calling mode. In receiving mode, mobile phone needs a lower amount of energy to be connected, while operating in calling mode oscillations of the emitted electric field increases. All ourAlteration of Enzymic Electron Transfer Reactions Induced by Microwaves... 13 experiments were carried out operating in receiving mode by dialling from a remote phone to the phone positioned at 5 cm far from the enzyme solution. As soon as the phone we utilized received a call, the emitted intensity of electric field rapidly increased in the first 10 s, reaching a maximum of 9 V m'', before decreasing to a lower standby asymptotic value of about 3 Vm". In this context, we carried out experimental kinetic measurements by comparing the enzymatic activity of native protein to aliquots of the same solution exposed 20 minutes and during the enzymatic reaction. Experimental measurements were made on native and irradiated before samples of the same enzyme solution to verify whether irradiation induce alterations on protein structure. Collecting kinetic data in absence and in presence of MWs we revealed as irradiation perturbs the reaction mechanisms. Our studies were made using bovine lactoperoxidase (LPO), ascorbate oxidase (AO) and laccase (LAC). Lactoperoxidase (LPO, EC 1.11.1.7) is a mammalian enzyme which is the product of exocrine gland secretion, e.g. into milk, saliva, and tears [6-8]. Bovine LPO consists of a single polypeptide chain containing 612 amino acid residues. The amino acid sequence is known and the molecular weight is approximately 78 kDa. LPO is a basic protein having an isoelectric point of 9.6 and it has about 10% of carbohydrate, localized on five potential N- glycosylation sites. Its secondary structure is composed of 23% a-helices, 65% B-sheet and 12% unordered structure. Figure 2 shows the Lineaweaver-Burk plots obtained following experimental procedure described above to perform kinetic measurements of guaiacol oxidation by hydrogen peroxide (H202) catalyzed by LPO [9]. The kinetic parameters KM, VM and kCAT, which are summarized in Table 1 were calculated from the intercept and the slope of the linear ‘fit. For native LPO these parameters are significantly lower than those of the irradiated samples. This difference is more marked in the case of LPO solution irradiated during the measurements, proving that exposure has considerably modified the enzyme activity. The reaction proceeds with a very complex mechanism [10], but it can be simplified as follows: +, wre H;02 —— LPO (FelV"") + H30 2 . 1 LPO (Fe "") + GUA—S-5 LPO (FeV) + GUA 2 LPO (Fe!Y) + GUA—*4-5 LPO (Fel) + GUA’ Table 1. Kinetic activity parameters of lactoperoxidase samples calculated from the slope and intercept of the straight lines depicted in Figure 1 Sample Ku @M) Vn (UM #5) Kear ©) Native 603+ 13 5.5403 180980 Irradiated before 203341 12.106 3980 + 199 Irradiated during 14250+ 285 6943 22697 + 113514 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri 20 1S 10 intensity of electric field (V/m) 0 t® Figure 1. EMF emission of the mobile phone recorded with PMM 8053 analyzer in both receiving (black histograms) and calling (grey histograms)mode [3]. 1.410" Fy 1825504 110.08" R= 0.99194 y = 82985 + 168.63x R = 0.99673 y= 14472 + 206.18x R= 0.98034 1.2:10° ~ 110° native < irradiated before = sigt tthe Feaction 5 irradiated during the reaction 610° 1000 ©2000» 3000» 4000» 5000-6000 1/[guaiacol] (M') Figure 2. Lineweaver-Burk plot of lactoperoxidase samples. Figure shows the plot of the inverse of initial reaction rate (1/v) versus the inverse of substrate concentration (1/{S]). [LPO]= 1.49x10°M, PBS 0.1 M (pH = 7.2), T = (25.0£0.1) °C. Data have been obtained spectrophotomerically by recording the absorbance at 470 nm of reaction product (tetraguaiacol).Alteration of Enzymic Electron Transfer Reactions Induced by Microwaves... 15 where Fe'Y"" and GUA" represent the free radical species of iron and guaiacol respectively. The final product of the reaction is tetraguaiacol, obtained from guaiacol radicals (GUA") combination. The kinetic equations which describe this process are: / d [Gua] — _Vy [Gua] °) dt [Gua] +K,, where: Vu = Kear[Enz], ; 8) aKalko+ ks) _ hea , Kako 4 Ki(kytky) ky ky(ky+ky) K3ky 6) koap = —34 ‘CAT (kj+k,) Kinetic measurements were collected spectrophotomerically at the tetraguaiacol absorption wavelength (470 nm). Data obtained demonstrated that exposure alters the kinetic parameters, probably modifying either conformational features of the enzyme active site or interfering with free radical intermediates species. To obtain further evidences, a set of electrochemical experiments (cyclic voltammetry) were carried out (Figure 3). 0 2 4 LPO native LPO irradiated LPO native + HO, LPO irradiated + HO, Iq@a) 0.6 0.4 -0.2 0 02 0.4 Potential (V vs Ag/AgCl) Figure 3. Voltammograms of the LPO enzymatic activity experiments [LPO] = 2.93x10°M, [H:0:] = 3.20x10"M, PBS 0.1 M, (pH = 7.2), T = (25.0+ 0.1) °C.16 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri 3000 = 4000 "5 , 3 0 & 2 z irradiated = -4000 5 2 -8000 200 208 216 224 232 240 248 256 A (am) Figure 4. Far-UV CD spectra of lactoperoxidase samples; the spectra were normalized with peptide concentration. [LPO]= 6.11x10-7M, PBS 0.1 M (pH = 7.2). The voltammograms were recorded in the range of potential where electron transfer between LPO (in solution) and gold electrode does not occur. In this case, reproducible and superimposable measurements of both native and irradiated samples (red and black curves) have been obtained. This result point out that irradiation does not enable altered protein to give rise electron transfer with electrode. Conversely, in the presence of H2O», the increase of the cathodic current (in the range between -0.25 V and -0.60 V) arises from the reduction of HO, catalyzed by LPO. In this case, the electrochemical measurements with native (curve blue) and irradiated enzyme (curve green) provide different trend in the voltammograms, proving that the increase of LPO catalytic activity is in direct relationship with conformational changes induced by MWs. Indeed, circular dichroism spectra (CD), recorded in peptidic bond absorption range (190-260 nm) (Figure 4), demonstrate that secondary structure of the enzyme doesn’t change up to 20 minutes of continuous irradiation. On the contrary, steady-state fluorescence measurements (Figure 5) show that irradiation provokes modification of the conformational states of tryptophan residues. In the LPO structure, tryptophans are placed in the proximity of the enzyme active site as schematically represented in Figure 6. This picture shows heme, proximal and distal His and the Trp residues placed close to the prosthetic group drawn using the crystallographic data extracted from Protein Data Bank (PDB) facility. Fluorescence measurements confirm that the difference found in the cyclic voltammetry measurements can be attributed to significant modification of LPO active site induced by mobile phone emission. After 20 minute of irradiation, LPO structural changes are irreversible and consistent with a new thermodynamically stable structure of the enzyme, providing reproducible spectroscopic and kinetic data. However these results do not explain the dramatic variation observed in kinetic measurements carried out exposing the enzyme- substrate mixture during the reaction. In this case, Km, Vm and kcar values (see Table 1) change of 1-2 orders of magnitude merely because the system was exposed to the mobileAlteration of Enzymic Electron Transfer Reactions Induced by Microwaves... pg phone during the re: ction development. This effect gives a further demonstration that pulsed and variable EMFs alter the mechanism of electron transfer processes [10-11]. 700 600 500 400 r 2802m (ua) 300 200 100 350 300 250 rot 412 am, 200 T(ua.) 150 100 50 native native /*\ irradiated 500 400 450 500 2 (nm) 550 600 650 700 % (am) (b) Figure 5. Steady-state fluorescence spectra of lactoperoxidase samples recorded exciting sample at 280 nm (aromatic residues absorption) (a) and exciting at 412 nm (active site absorption) (b). Both in the cases irradiation determines fluorescence quenching (dotted line) showing that EMFs provoke a shift of Trp residues from the inside towards the solvent and an alteration of active site conformation. [LPO] = 6.11x107M, PBS 0.1 M. (pH = 7.2), T = (25.0 + 0.1)°C.18 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri Trp 47 ID PDB : IMYP Figure 6. “Ball and sticks” representation of LPO active site. In the picture the heme group is represented in red, His in CPK colour, while Trp 32, 436, 477 are blue. At this stage, we wondered whether data obtained for LPO may be extended to other metallo-proteins. The question was if enzymes with different amino-acid sequence and active site can be modified by MWs irradiation. For this purpose, we studied the influence of mobile phone emission on two copper active site enzymes: ascorbate oxidase (AO) and laccase (LAC). These enzymes are member of the multicopper oxidases family, which catalyze the oxidation reaction of suitable substrates with oxygen through electron transfer mechanism. They have got the same active site, containing four tightly bound copper ions distributed in three different redox sites, namely type-I, type-2, type-3 and classified on the basis of their spectroscopic features. In particular, they are composed of one type-I (T1) copper, which is the site of substrate oxidation (E° = +790 mV), of one type-2 (2) and two type-3 (T3) copper ions arranged in a trinuclear cluster. The electrons yielded by substrate oxidation are transferred to trinuclear cluster and used to reduce molecular oxygen to water. This reaction occurs by outer-sphere electron transfer mechanism, through electronic tunnelling mediated by aromatic amino acids residues [12]. The effects of irradiation were carried out using the same experimental procedure we adopted for LPO. Figure 6 shows the Lineaweaver-Burk plot for the enzymatic reaction of ascorbic acid oxidation by oxygen. Kinetic data were collected by means of electrochemical measurements (chronoamperometry) using the same AO solution, either irradiating the enzyme before for 20 minutes (red line) or under continuous irradiation during the reaction (blue line). The kinetic parameters (Ky, Vin and Kear) obtained are summarized in Table 2, where a significant alteration of the oxidation process, induced by exposure of the reaction bulk to the MWs, appears evident. These parameters, calculated from the slopes and the intercepts of the linear plots (Figure 7) reveal that a general inhibition mechanism was induced, since V, and Kear Values are lower than those of native sample. Somewhat surprisingly, the enzyme sample irradiated before to be employed in the catalytic reaction, exhibits a peculiar behaviour, showing an evidentAlteration of Enzymic Electron Transfer Reactions Induced by Microwaves... 19 deviation from Michaelis-Menten mechanism. Data do not give a linear Lineaweaver-Burk plot, indicating that the reaction mechanism is deeply modified. Our opinion is that MWs probably induce conformational modification of the AO catalytic site giving rise to a number of different conformers. Each of them is characterized by peculiar catalytic activity due to changes in the metal ion environment, which may concern non reproducible enzymic activity. This last observation is corroborated by steady-state fluorescence measurement of the intrinsic tryptophan emission spectra showed in Figure 8 (panel a), as irradiation determines a quenching of tryptophan emission intensity of about 9%. Commonly, the decrease of the intensity is associated to a conformational change of Trp residues which are shifted from the protein inside towards the solvent [12]. Furthermore, the fluorescence spectrum of type-3 copper ions (panel b Figure 8), recorded exciting at 330 nm, shows an increase of the intensity of about 15%, confirming that MWs induce structural changes in the AO active site. 7 310 y= 12909 + 13.326x RK =0.99027 y= 11097 + 19.892 x R= 0.98603 2.510" native = irradiated before - irradiated during & R210" E 1.5 10° 110' 0 100 200 300 400 500 600 700 800 [ascorbic acid] (M') Figure 7. Lineweaver-Burk plot of ascorbate oxidase samples. Figure shows the plot of the inverse of initial reaction rate (1/v) versus the inverse of substrate concentration (1/[S]). [LPO]= 3.60x10°M, PBS 0.1 M (pH = 7.2), T = (25.0+0.1) °C. Data have been obtained using an electrochemical techniques (chronoamperometry) by recording the oxidation of ascorbic acid (substrate) at 700 Mv. Table 2. Kinetic activity parameters of ascorbate oxidase samples calculated from the slope and intercept of the straight lines depicted in Figure 6 Sample Km (uM) Vn (UM #8") Kear (") Native 179270 9043 1782469 Irradiated before non-detectable non-detectable non-detectable Irradiated during 1032+ 50 1122 152544220 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri 400 native irradiated 350 300 250 (ua), 200 150 100 50 300 350 400 450 500 700 /7\ native \ irradiated I(ua,) 0 340 360 380 400 420 440 460 480 500 2 (am) ) Figure 8. Steady-state fluorescence spectra of ascorbate oxidase recorded exciting sample at 295 nm (a) (tryptophan residues absorption) and exciting at 330 nm (type-III copper (b). Irradiation determines fluorescence quenching (dotted line) exciting at 295 nm showing that EMFs provoke a shift of Trp residues from the inside towards the solvent and a fluorescence increase (dotted line) exciting at 330 ‘nm showing an alteration of active site conformation due to MWs. [AO] =1.65x107M, PBS 0.1 M (pH = 7.2), T= (25.04 0.1)°C. As it has already found for LPO, also in this case, we observed a modification of enzyme active site without any modification of secondary structure (see far UV CD spectra, Figure 9).Alteration of Enzymic Electron Transfer Reactions Induced by Microwaves. 21 4000 ~ 2000 5 0 J = -2000 3 ge 4000 irradiated 3 = -6000 5 native © -8000 -110" -1.2 108 ee 200 210 «220230240 250260 2 (nm) Figure 9. Far-UV CD spectra of ascorbate oxidase samples; the spectra were norma concentration. [AO]=1.65x10"M, PBS 0.1 M (pH = 7.2). ed with peptide Figure 10 shows the AO active site and Trp residues surrounding copper metal ions which are subject of structural modifications. Figure 10. Schematic representation of AO active site got by crystallographic data (ID PDB:1A0Z). Copper ions are pictured as green balls and Trp residues surrounding the active site are reported as “ball and stick” model. Laccases and AO have got very similar active sites, but different protein structures originated from different amino acid sequences. Generally, laccases have a single2 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri polypeptidic chain, while AO is a dimer. To carry out comparative evaluation of the MWs effects, we chose the laccase extracted from Agaricus Bisporus (LAC) because in water solution it is a dimer, as well as AO. The first results we obtained was that irradiation did not change secondary and the tertiary structure of LAC. Far-UV CD and steady-state fluorescence spectra of native and irradiated before (20 minutes) LAC revealed that any structural modification occurred to the protein samples. These experimental evidences are shown in Figures Ila and 11b respectively, where both.CD and fluorescence spectra are neatly superimposable. (mdegree*M"*cem") native irradiated 200° «210220 230 240250 2 (am) (a) native irradiated I(wa) 320 360 400-440 480-520 2 (am) (b) Figure 11. Far-UV circular dichroism (a) and Steady-state fluorescence spectra (b) of laccase samples recorded exciting sample at 280 nm (a) (aromatic residues absorption). [LAC] =9.70x10°M, PBS 0.1 M (pH = 7.2), T = (25.0 + 0.1)°C. CD spectra were normalized with peptide concentration.Alteration of Enzymic Electron Transfer Reactions Induced by Microwaves... 23 0.003 0.0025 © s 8 native irradiated before the reaction 0.0015 (mM*s") 0.001 irradiated during the reaction 0.0005 Oo 1 2 3 4 5 6 7 [catechol] (mM) Figure 12. Michaelis-Menten plot of laccase samples. [LPO]= 2.37x107M, PBS 0.1 M (pH = 7.2), T= (25.00.1) °C. Data have been obtained spectrophotomerically by recording the absorbance at 450 nm of reaction product. Table 3. Kinetic activity parameters of laccase samples calculated from the parameters of Michaelis-Menten fit depicted in Figure 12 Sample Km (uM) ‘Vim (uM *s-1) KCAT (s-1) Native 1500+ 100 2.7£0.1 u2to0.1 Irradiated before 1300+ 100 28t0.1 iusto. Irradiated during 3600+ 400 4.1t03 16.9£0.3 Figure 13. Molecular structure of phenol red.24 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri 08 0.45 E non-irradiated & ms irradiated < 0.35 0.3 0 100 200 300 ©6400 ©= 500600 t(s) Figure 14, Phenol red in TRIS buffer with and without irradiation with mobile phone ([phenol red]) = 1.05x10°M, TRIS buffer 0.04 M (pH = 8.3), T = (25.0 + 0.1) °C. Kinetic measurements of the LAC enzymic activity provided unchanged parameters which confirmed the lack of effects due to MWs exposure. On the contrary, the same reaction carried out under MWs field is significantly modified. These results are shown in Figure 12 and the kinetic parameters Ky, Vm and Kear, calculated by means of the best fit of the Michaelis — Menten plot, are summarized in Table 3. The findings that a kinetic effect has been obtained irradiating the reaction bulk put in evidence that the electromagnetic field interferes with the reaction mechanism mediated by free radical intermediate species. To understand the difference between AO and LAC catalytic activity, we considered that despite the proteins are both dimers with the same active site, they have got a different overall dipole momentum (fio = 1156 Debye, pac = 629 Debye). It seems reasonable to suppose that the molecular overall dipole momentum plays an important role in determining conformational changes in the protein structure. On the other hand, also LPO has a high dipole momentum (jt = 1055 Debye) and it shows structural modifications as well as AO. In the case of LAC there is instead a close relationship between protein structural stability and low value of dipole momentum. Nevertheless, the hypothesis that the EMFs could induce a “instantaneous” thermal effect should be considered, since an increase of temperature due to irradiation can explain the observed changes of the enzyme activities. Temperature fluctuations in the kinetic experiments can be determined using the TrisHCI - Phenol Red method [13]. This procedure represents a “chemical thermometer” because it exploits the sensitivity of the TRIS buffer to the temperature. A change of temperature varies the buffer pK and, in turn, the solution pH which can be relieved measuring the optical density of a suitable concentration of the indicator (see Figure 13). Phenol red has a strong absorbance band at 558 nm (red) and it shows a pH dependent dissociation equilibrium: Dy Dy + 1Alteration of Enzymic Electron Transfer Reactions Induced by Microwaves... 25 where Dy and Dk are the phenol red indissociated and dissociated form respectively. Figure 14 shows the Assz am VS time plots of non-irradiated (black) and irradiated samples (red) phenol red in TRIS buffer solution. It appears evident that, since any absorbance fluctuation has been recorded, no thermal effects have been reviled up to 600 seconds of mobile phone irradiation, Conclusion In this chapter the experimental data prove that the mobile phone emission alters the features and the catalytic activities of metallo-enzymes. These phenomena have been attributed to the electrostatic interactions between the oscillations of the electric field associated to MWs and charge distribution in the protein structure. It has been also observed that electron-transfer mechanisms, as well as free radical intermediates species, are sensitive to the “external” fluctuation of electromagnetic field. Similar structural modifications have been already observed in protein solutions containing low concentration of denaturating agents, such as guanidine or urea [12]. Among different possible protein denaturated states, molten globule has been defined as such a thermodynamically stable feature occurring when tertiary protein structure changes without secondary structure modifications [12]. The spectroscopic data we recorded demonstrate that mobile phone emission can induce structural changes ascribable to a molten globule conformation. Far-UV CD spectra gathered in the peptide absorption range show that irradiation does not affect secondary structure of the enzyme, while evidence of tertiary structure modification is revealed by the decrease of tryptophan fluorescence. Despite these results cannot be used to conclude that the mobile phone emission can lead to hazardous effect for the human health, data here showed may represent a useful starting point to improve our knowledges on the effects of the MWs exposure. The use of highly purified protein samples represents a useful tool to obtain reliable and reproducible experimental data even using a widely variable radiation source. References [1] Maneinelli F., Caraglia M., Abbruzzese A., d’Ambrosio G., Massa R., Bismuto E., J. Cell. Biochem. 2004, 93, 188. [2] Copty A. B., Neve-Oz Y., Barak I., Golosovsky M., Davidov D., Biophys. J. 2006, 91, 1413. {3] Barteri M., Pala A., Rotella S., Biophys. Chem. 2005, 113, 245. [4] Malaric K., Bartolic J., Malaric R., IMTC 2004, Instrumentation and Measurement Technology Conference, Coro, Italy, 18-20 May 2004. [5] _ van Holde K.E., Johnson W.C., Shing Ho P., Principles of Physical Biochemistry, ed. by Prentice-Hall Inc., Upper Saddle River, New Jersey, USA, 19! [6] K.M. Pruitt, J. 0. Tenovuo, , The Lactoperoxidase System: Chemistry and Biological Significance, ed. by Marcel Dekker, Inc., New York, USA, 1985. [7] _ J. Everse, K. E, Everse, M. B. Grisham, Peroxidases in Chemistry and Biology, ed. by CRC Press, Inc., Boca Raton, FL, 1991, Vol. I, pp 143-180.26 Roberta De Carolis, Fiorenzo Marinelli and Mario Barteri [8] _E.L. Thomas, P. M. Bozeman, D. B. Leam, Lactoperoxidase. New Recognition of an “Ola” Enzyme in Airway Defenses, ed. by CRC Press, Inc., Boca Raton, FL, 1991 Vol. 1, pp 123-142. [9] Doerge D.R., Divi R.L. , Churchwell M.I., Anal. Biochem. 1997, 250, 10. [10] M.Blank, L. Soo, Bioelectrochemistry 2003, 61, 93. [11] M. Blank, L. Soo, J. Cell. Biochem. 2001, 81, 78. [12] K.E. van Holde, Principles of Physical Biochemistry, ed. by Prentice-Hall, Inc., Upper Saddle River, New Jersey, USA, 1998, pp 28-42. [13] B. Chance, A. C. Maehly, Methods in Enzymology, ed. by S.P. Colowick and N.O.. Kaplan, Academic Press, New York, USA, 1964, Vol. II, pp 764.