Amperometric monitoring of redox activity in intact, permeabilised and lyophilised cells of the yeast Hansenula polymorpha

Electrochemistry Communications 9 (2007) 1480–1485 www.elsevier.com/locate/elecom

Amperometric monitoring of redox activity in intact, permeabilised and lyophilised cells of the yeast Hansenula polymorpha Maria Khlupova a, Boris Kuznetsov a, Mykhailo Gonchar b, Tautgirdas Ruzgas d, Sergey Shleev a,c,d,* b

a Laboratory of Chemical Enzymology, Bach Institute of Biochemistry, 119071 Moscow, Russia Department of Cell Regulatory Systems, Institute of Cell Biology, Drahomanov Street 14/16, 79005 Lviv, Ukraine c Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden d Biomedical Laboratory Science, Faculty of Health and Society, Malmo¨ University, SE-205 06 Malmo¨, Sweden

Received 15 January 2007; received in revised form 5 February 2007; accepted 14 February 2007 Available online 20 February 2007

Abstract An effect of permeabilisation and lyophilisation of the yeast cells Hansenula polymorpha on their electrochemical behaviour in the presence of mediators, substrates (formaldehyde, glucose, methanol, ethanol), and cofactors (NAD+, NADP+, NADH, NADPH, glutathione) has been studied. Two amperometric techniques differing in the cell immobilisation methods were applied. The cells of a wild strain (356) and mutant strains (C-105 and KCA 33) of the yeast, grown in the presence of glucose or methanol, were used in the experiments. The intact cells revealed the highest reduction rates of mediators, 2,6-dichlorphenolindophenol (DCIP) and 2,4-benzoquinone (BQ), as measured by amperometry. The addition of formaldehyde significantly enhanced the response, if the cells were grown in the presence of glucose. The permeabilised cells showed the lowest current level in the presence of DCIP and BQ and no response to the addition of formaldehyde and NAD+. However, the addition of NADH gave significant current surge. All these phenomena imply that the permeabilised cells lost cofactors and the activity of dehydrogenases producing NADH, but they remained the activity of NADHubiquinone oxidoreductase and of some components of the electron transport chain. The electrochemical behaviour of the lyophilised cells shows they are heterogeneous. The partial degradation of the outer membrane of the cells after their lyophilisation was electrochemically confirmed. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Yeast Hansenula polymorpha; Intact, permeabilised and lyophilised cells; Amperometry; Mediators

1. Introduction Electrochemical activity of living cells, i.e., generation of current at cell modified electrodes in the presence of a mediator, can be considered as an indicator of their metabolic activity. Nonliving cells are electrochemically inactive [1–3]. The changes in the redox activity of the cells mean *

Corresponding author. Address: Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Tel.: +46 46 222 8191; fax: +46 46 222 4544. E-mail addresses: [email protected], [email protected] (S. Shleev). 1388-2481/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2007.02.011

that the electron transport in the biological system and the physiological state of living cells has been modified [4]. The electrochemical studies of the redox state of living cells might be useful for monitoring of different pathophysiological states of the cells, such as an oxidative and nutrient stresses, effect of cytotoxic, mutagenic, and carcinogenic preparations [4–6]. Many organic electroactive substances, e.g., 2,6-dichlorophenolindophenol (DCIP), 2,4-benzoquinone (BQ) and other quinone derivatives, are hydrophobic enough and capable to permeate through the membrane and, thus, act as electron transfer mediators between intracellular redox processes and electrodes. Moreover, combination of mediators, e.g., application of

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both organic and inorganic compounds together (K3[Fe(CN)6] and BQ or DCIP), gives a significant increase of the electrochemical response [7]. All these single mediators and double mediators systems are often used in ‘‘cell electrochemistry’’ nowadays [2,4,5,7]. They collect electrons from intracellular reducing agents produced by metabolic reactions. Components of the respiratory chain and FAD containing dehydrogenases are the most active in the electron-exchange reactions with mediators [3,7,8]. The function of different mediator systems as shuttles between an electrode and different cells, such as yeast, bacteria, and mammalian, is well described in the literature [2,7–9]. The respiratory chain of the mitochondrial inner membrane includes a proton-pumping enzyme, complex I, which catalyses the electron transfer from NADH to ubiquinone pool [10,11]. The mitochondria also contain several nonproton-pumping alternative NAD(P)H dehydrogenases. As known these reactions are the firsts in the chain of electron transport. Thus, the detection of intracellular redox reactions using a mediator is based on its reduction by the cytosolic and mitochondrial enzymes and electron-carriers catalysing electron transfer from NADH and NAD(P)H along the electron transport system. The goal of the present work was to elucidate the effect of the membrane permeability on the function of the electron transfer system and some dehydrogenases, as well as to clarify possible differences in the redox properties of intact, lyophilised, and permeabilised cells. Towards this end the amperometric monitoring of redox activity of intact, lyophilised, and permeabilised cells of different strains of the methylotrophic yeast Hansenula polymorpha, which differ in metabolic characteristics [12–14], was performed using two different electrochemical techniques and several redox mediators.

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The wild type and mutant strains of the thermotolerant methylotrophic yeast H. polymorpha constructed in Institute of Cell Biology (Lviv, Ukraine) were used, namely intact cells of the wild type strain 356, mutant strain KCA 33 (gcr1 catX), and permeabilised cells of a mutant strain C-105 [13,14]. The strain H. polymorpha KCA 33 has impairment in glucose catabolite repression of alcohol oxidase (AOX) synthesis, it is catalase-defective, and it has the ability to perform constitutive synthesis of AOX in a glucose-containing growth medium, in contrast to the wild type strain 356 unable to synthesise this enzyme in the presence of glucose. 2.3. Cultivation of the yeast H. polymorpha and preparation of lyophilised and permeabilised cells Cells of H. polymorpha (any strain) were cultivated in flasks on shaker (200 rpm) at 30 °C to the middle of the exponential growth phase (24 h) in the medium containing (g/L): glucose – 10; (NH4)2SO4 – 3.5; KH2PO4 – 1.0; MgSO4 – 0.5; CaCl2 – 0.1; yeast extract – 3.0. The pH of the medium was 5.5. Permeabilised cells of the H. polymorpha C-105 yeast were prepared as described herewith [15]. Briefly, yeast cells at a concentration of 4–5 mg/ml were treated with 0.1% digitonin in 50 mM K-phosphate buffer, pH 7.0 for 15 min at 30 °C on shaking periodically. The cells were washed twice with the initial buffer, separated by centrifugation at 1000 rpm at 4 °C, lyophilised, and kept at 15 °C. Before each experiment lyophilised yeast cells were re-suspended in 0.1 M K-phosphate buffer, pH 7.0 at the concentration of 0.8 mg/ml and allowed to swell during 30 min at 37 °C. Just after the swelling the suspension of cells was used in the electrochemical experiments. 2.4. Amperometric studies

2. Experimental 2.1. Chemicals K2HPO4, glucose, yeast extract, methanol, and ethanol were from ‘‘Reachim’’ (Moscow, Russia). Digitonin, CaCl2, KCl, MgSO4, (NH4)2SO4, and KH2PO4 were from ‘‘Merck’’ (Darmstadt, Germany). Tris, paraformaldehyde, succinate, 2,6-dichlorophenolindophenol (DCIP), 2,4benzoquinone (BQ), 1,2-naphthoquinone, K3[Fe(CN)6], reduced and oxidised glutathione, NAD+, NADP+, NADH, and NADPH were from ‘‘Sigma’’ (St. Louis, MO, USA). Buffers were prepared using double-distilled water. 2.2. Materials and cells The solution of formaldehyde (1 M) was prepared by hydrolysis of the corresponding amount of paraformaldehyde (300 mg; 10 ml water) in sealed test-tubes placed in water bath for 3 h at 100 °C.

2.4.1. Measurements by using a glassy-carbon electrode pressed to the filtered cells The electrochemical measurements were performed by using glassy-carbon electrode according to [3,4]. The suspension of cells of H. polymorpha (0.5 ml) was filtered through Schleicher & Schull filter (Germany, diameter 7 mm, with pore diameter 0.45 lm). The filter was placed on the bottom of the electrochemical cell and the working glassy-carbon electrode (Bioanalytical Systems, West Lafayette, IN, USA) recorded to investigate whether proofs of direct heterogeneous was lightly pressed to the filter together with the salt bridges of the Ag/AgCl/KClsat reference electrode, and auxiliary platinum electrode. Then 0.5 ml of the K-phosphate buffer containing 0.2 mM mediator with or without cell’s substrate (formaldehyde) was introduced to the yeast cell sample being on the filter. The potential of the electrode was measured by open circuit for 5 min using BAS CV-50W Electrochemical Analyser with BAS CV-50W software v. 2.1. Then the potential of 250 mV was applied and the current was recorded for

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200 s. It was established previously that alive cells retained intact at applied potential of +250 mV vs. Ag/AgCl/KClsat (ca. 450 mV vs. NHE) during the time of the experiment [4]. This method is more suitable to use while prolonged monitoring of the dynamics of cell growth or cell activity. But the technique does not allow realising any preparation additions to the sample during the experiment. 2.4.2. Amperometric studies of redox activity of immobilised cells using screen-printed platinum electrode Principal scheme of measurement of intracellular redox reactions using 2,6-dichlorophenolindophenol (DCIP) as a mediator as well as putative mechanism of DCIP reaction cycle in the intact cells were described previously [16]. Intact, lyophilised, and permeabilised yeast cells were immobilised on the top of platinum band electrode in a Ca2+-alginate gel as described herein [16]. For this aim, 5 mg of the cells were suspended in 0.3 ml of 10 mM Tris–succinate buffer, pH 7.0. After that, 15 ll of 2% sodium alginate was added to the 15 ll of the suspension of yeast cells. The electrode was coated with 15 ll of cellalginate suspension by the sampler. The excess of cellalginate was removed by a tip. The calcium alginate gel was formed after immersing the coated electrode in solution of 100 mM CaCl2 for 2 min. Measurements were carried out immediately after the cell immobilisation was completed. To maintain the integrity of the gel the measurements were performed in the buffer containing 10 mM CaCl2. One electrode modified with the layer of the gel containing immobilised cells was used for one series of experiments. Electrochemical measurements were carried out at a fixed potential of +270 mV in an electrochemical cell under aerobic conditions, provided by vigorous stirring. Eightchannel screen-printed electrode from ‘‘BVT Technologies’’ (Brno, Czech Republic) was used. It composed of two elements: working Pt electrodes and combined reference–counter Ag electrodes. Yeast cells were immobilised on this electrode. The electrode was placed then into the electrochemical cell containing 15 ml of buffer solution (10 mM Tris–succinate, pH 7.0). Measurements started after the steady-state current became established. Then 2 ml of buffer containing mediator DCIP was added to a final concentration of 0.1 mM by 200 s. After the resulting response had reached a steady state, the measurement was continued by the addition of reduced and oxidised cofactors: NAD(P)H, NAD(P)+, reduced or oxidised glutathione, and substrates: formaldehyde, methanol, and ethanol. This method is well suitable for real time control of cell activity by addition of different preparations during the experiment. The standard deviations have been calculated from results of three independent experiments and were found to not exceed 20% in all electrochemical measurements, when the same cell culture was used, whereas the variation of the cell activity for three different sowing samples was found to not exceed 50%.

3. Results and discussion The amperometric responses of intact yeast cells of H. polymorpha KCA 33 and permeabilised yeast cells of H. polymorpha C-105 immobilised on the glassy-carbon electrodes in the presence and absence of different mediators are presented in Table 1. As seen from the table insignificant current outputs were obtained in the absence of mediators for both intact and permeabilised yeast cells, whether formaldehyde, a cell substrate, was added to the solution or not. Obviously, in the absence of mediators significant current from the electrode functionalised by cells might be obtained only if direct electron transfer between the glassy-carbon electrode and the yeast cells is realised. Native cells are known to excrete no redox active compounds, which can significantly affect electrochemical signal [1,17]. Even if direct electron transfer reactions were recently shown for some living cells containing unique microbial nanowires (pili) or outer-membrane c-type decaheme cytochromes [18,19], the yeast cells of H. polymorpha are not able directly communicate with an extracellular solid matrix. Nevertheless, in the absence of mediator the appreciable currents are revealed for both intact and permeabilised cells. These currents can be considered as a result of charge leakage through the cell membranes since the intact state of the cells is maintained. On contrary, the currents observed by voltammetry are similar to ones of the insulation breakdown, since the cells and its membranes become destroyed when the applied scanning potential exceeds 500–600 mV during measurement [19]. In the presence of hydrophobic mediators very high currents were observed, when intact cells were immobilised at the electrode. Hydrophilic inorganic mediator, K3[Fe(CN)6], was inefficient as it is incapable to penetrate through the yeast cell membrane. It enhances rather the electron leakage resulting in the low increase of the current. The most efficient mediator proved to be BQ due to its optimum solubility in both the membrane medium and water, and also due to its high redox potential equal to 270 mV. Compound 1,2-naphthoquinone is too hydrophobic and only slightly soluble in water that decreases significantly its efficiency as possible redox mediator. In the absence of formaldehyde, the redox reactions seem to occur owing to the endogenous reducing agents. In the presence of formaldehyde the intracellular alcohol dehydrogenase realises synthesis of NADH that makes the electron transport chain to function more intensively. Therefore, formaldehyde caused high currents, if the intact cells and hydrophobic mediators, DCIP or BQ, were employed (Table 1). The electron transport chain in the permeabilised cells does not act entirely for lack of coenzymes, NAD, NADP, and other low-molecular compounds, e.g. glutathione. Most likely, they were washed off during the preparation of the permeabilised cells since their outer membranes became transparent for these compounds. The current response of permeabilised cells was by 10

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Table 1 Amperometric response (in nA) of intact and permeabilised yeast cells of H. polymorpha C-105 immobilised on the glassy-carbon electrode in the presence and absence of different mediators Cells

Conditions

Without mediator

DCIP

K3[Fe(CN)6]

BQ

1,2-Naphthoquinone

Intact

FA Without FA

12 14

266 234

48 48

2956 1682

231 675

Permeabilised

FA Without FA

6 5

35 35

76 69

168 172

105 116

Notes: Cells’ density on the filter, 0.2 mg/cm2; formaldehyde concentration, 1.8 mM. The S.E.M. has been calculated from three independent experiments and did not exceed 20%. The measurements were performed in accordance with [4].

times less than the response of the intact cells in the presence of both DCIP and BQ. Moreover, the current output did not increase after the addition of formaldehyde. Possible explanation of these facts can be found if one supposes that the some oxidases are maintained inside the permeabilised cells. Then the mediators function as electron acceptors. But alcohol oxidase (AOX) catalysing oxidation of formaldehyde has, unlike the other oxidases, a very high rate with oxygen, its natural electron acceptor. Therefore, the AOX rate with oxygen is much higher than that with DCIP and BQ [17]. For this reason no increase of the current was observed by addition of formaldehyde. The second technique, which employs the screenprinted platinum working electrode modified with the immobilised yeast cells, allowed us to elucidate some additional details. Fig. 1a shows the behaviour of the immobilised intact thermotolerant methylotrophic yeast cells H. polymorpha, strain KCA 33. The electrode was immersed into the stirred buffer solution and the chronoamperogram current was first insignificant. The addition of DCIP at 200 s stimulated the current increase up to 330 nA (Fig. 1, curve 1). The high current was caused by function of the different dehydrogenases and possibly some other redox proteins of the electron transport chain. The mediator DCIP is able to accept electrons from all of these components of living cells. Endogenous substrates of cells were used in the reactions. Further addition of formaldehyde to this solution was made by 550 s. The pronounced current development followed the formaldehyde addition, which amounted to 250 nA. These results definitely imply that the intact cells have, first, the porins in the outer cell membrane, which provide the pass of formaldehyde inside the intact cells. Second, they contain very active cytosolic and mitochondrial alcohol dehydrogenases catalysing electron transfer from the different substrates to NAD(P)+. For example, one of the well-known cytosolic NADHproducing enzymes from the methylotrophic yeast is formaldehyde dehydrogenase (FD, EC1.2.1.1), which is, besides NAD, glutathione-dependent contrary to bacterial enzyme. This enzyme is able to oxidise formaldehyde, exogenously added or produced inside the cells, in alcohol oxidase-catalysed reaction of methanol oxidation with concomitant production of NADH in the presence of glutathione:

GSH þ CH2 O ¼ GS  CH2 OH

ð1Þ

GS  CH2 OH þ H2 O þ NADþ ¼

HCOOH þ NADHðHþ Þ þ GSH

ð2Þ

Another NADH-producing enzyme, involved in methanol metabolism, is formate dehydrogenase (EC 1.2.2.1), which oxidises formic acid, product of the reaction (2), to carbon dioxide: HCOOH þ NADþ ¼ CO2 þ NADHðHþ Þ

ð3Þ

The reactions catalysed by cytosolic and mitochondrial dehydrogenases give additional growth of current. If one added NADH or NADPH instead of formaldehyde the current do not increase (Fig. 1a, curve 2). This means the membranes of the intact cells are impermeable for these coenzymes and this fact is generally recognised. Fig. 1b demonstrates the results of the experiments with permeabilised cells H. polymorpha C-105. The current surge as result of the DCIP insertion was 10 times less than with intact cells. Further addition of the substrates, viz. formaldehyde, methanol, and ethanol, as well as different cofactors, such as NAD+, NADP+, reduced and oxidised glutathione, into the electrochemical cell with screenprinted platinum working electrode modified by permeabilised yeast cells did not affect the steady-state current of the electrode (Fig. 1b). This means the permeabilised cells have lost the activity of the most part of cytosolic FAD containing dehydrogenases capable to reduce NAD(P)+ in spite of the fact that another cytosolic enzyme, AOX, preserved its activity enough well under the same conditions [16]. This distinction can be explained by a significant difference in molecular sizes of cytosolic FAD containing dehydrogenases and AOX: big octameric AOX molecules remain its intracellular location even after permeabilisation of the cells, contrary to much smaller molecules of dehydrogenases which can leak from the cells treated by digitonin. On the contrary, the addition of NADH and NADPH stimulated the significant growth of current (Fig. 1b, curves 1 and 2). This implies that the key mitochondrial NADH dehydrogenase (NADH-ubiquinone oxidoreductase, EC 1.6.5.3) and possibly some other oxidoreductases [20,21] do maintain the activity inside the permeabilised cells. The observed response can be only as result of reaction of DCIP with the reduced enzymes, with the reduced ubiquinone, or even with other components of the electron

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a

M. Khlupova et al. / Electrochemistry Communications 9 (2007) 1480–1485

1

600

500

Current, nA

FA 400

2

that for the intact and permeabilised cells. As seen in Fig. 2a the response on the DCIP addition (ca. 100 nA) was generally less than that for intact cells but higher than for permeabilised ones (cf. Figs. 1a, b, and 2a corresponding to the intact, permeabilised and lyophilised cells, respectively). This suggests the composition of the lyophilised cells is heterogeneous. A part of the cells is retained

300

a

NAD(P)H

250

1

200 200

100

DCIP

200

400

600

800

1000

Time, s

b

Current, nA

0 0

NADH

150

3 CH3OH NADH

100

140

1

50

120

DCIP

100

Current, nA

2

FA

C2H5OH

0 100

200

2

80

NADH NADPH Substrates

60

300

400

500

600

Time, s

b

150

FA 1

3

40

700

125 NAD(P)+ or glutathione

DCIP 20 0 0

200

400

600

800

1000

Time, s Fig. 1. Typical real-time current responses of yeast cells immobilised on the screen-printed platinum electrode. (a) Intact cells of the thermotolerant methylotrophic yeast H. polymorpha KCA 33 and (b) permeabilised cells of the mutant strain of the yeast H. polymorpha C-105. Composition of electrolyte: 10 mM Tris–succinate buffer (pH 7.0), 10 mM CaCl2, and 100 mM KCl. (a) Curve 1 – DCIP was added at 240 s and substrates (formaldehyde (FA), methanol (CH3OH), and ethanol (C2H5OH)) – at 500 s; curve 2 – DCIP was added at 200 s and NADH or NADPH – at 500 s. (b) Curves 1 and 2 – DCIP was added at 200 s, NADH (curve 1) and NADPH (curve 2) up to 1 mM were added at 520 s; curve 3 – DCIP was added at 200 s, substrates (formaldehyde (FA), methanol (CH3OH), and ethanol (C2H5OH)) – at 500 s with the following addition of NAD+ or NADP+ or glutathione up to 1 mM at 550 s.

transport chain because NAD(P)H is not oxidised both by DCIP and electrochemically at the potential of 250 mV. It is well known fact that after the lyophilisation the yeast cells remain intact. It was interesting to perform electrochemical investigation of the lyophilised cells of the yeast H. polymorpha C-105 and compare the results with

Current, nA

100

2

75

FA

50 DCIP

25

0 0

200

400

600

800

1000

Time, s Fig. 2. Typical current responses of yeast cells immobilised on the screenprinted platinum electrode. Electrolyte: 10 mM Tris–succinate buffer (pH 7.0), 10 mM CaCl2, and 100 mM KCl. (a) Intact lyophilised cells H. polymorpha C-105. Curve 1 – DCIP was added at 210 s, formaldehyde (FA) – at 340 s, and NADH at 425 s. Curve 2 – DCIP was added at 210 s, ethanol (C2H5OH) – at 360 s, and NADH – at 500 s. Curve 3 – DCIP was added at 210 s, methanol (CH3OH) – at 410 s, and NADH – at 500 s. (b) Wild type yeast cells H. polymorpha, strain 356. Curve 1 – cells were cultivated on the glucose-containing medium. Curve 2 – cells were cultivated on the methanol-containing medium. DCIP was added at 200 s and formaldehyde - at 520 s.

M. Khlupova et al. / Electrochemistry Communications 9 (2007) 1480–1485

intact and the other part of cells has damaged membranes. The conclusion is also supported by the following facts. A very little current output was observed from the cells, grown on the glucose-containing medium, towards formaldehyde, whereas significant increase of the current was found after the addition of NADH. In the presence of NADH strong increase of the current was observed for the lyophilised yeast, almost the same as for permeabilised cells (cf. curves 1 in Figs. 1b and 2a) suggesting partial degradation of the outer membrane of cells after their lyophilisation. However, a fraction of the cells is still intact, which was confirmed by the presence of the amperometric response towards formaldehyde and especially ethanol, but not methanol (Fig. 2a). The latest, in all likelihood, is related to another reaction of methylotrophic metabolism, catalysed by formaldehyde reductase (FR), which runs in the opposite direction if compared to formaldehyde reaction: þ

CH2 O þ NADHðH Þ ¼ CH3 OH þ NAD

þ

ð4Þ

Thus, substrate selectivity of the yeast cells can be explained by the following suggestions: (i) gcr1 mutation-caused genetic background (intact lyophilised cells H. polymorpha, mutant strain C-105), which affects on synthesis of FR by such manner that the activity of FR prevails causing the drop in current after addition of methanol (Fig. 2a); (ii) this mutation does not cause a serious effect on ethanol metabolism. It was shown in our recent papers that, to a first approximation, the current presented in Table 1 characterises the total cell’s metabolic activity [3,4]. Thus, it was interesting to investigate the metabolic activity of intact wild type yeast H. polymorpha depending on the cultural media. As can be seen from Fig. 2b the steady state current from the cells grown in the presence of methanol as carbon source was approximately two times higher compared to the yeast grown on the glucose-containing medium (40 nA vs. 21 nA; cf. curves 1 and 2). It is also important to note that the yeast H. polymorpha grown in the presence of glucose showed pronounced amperometric response towards glucose and ethanol, but not methanol, whereas the cells grown on methanol-containing medium showed quite pronounced amperometric response towards both alcohols and produced only very little steady-state current after glucose addition (data not shown). These data are in a full agreement with the peculiarities of methylotrophic metabolism regulation, when glucose causes catabolite repression of methylotrophic enzymes’ biosynthesis and methanol is the inducer of such enzymes [22]. Thus, the electrochemical analysis of the yeast cells by using mediators has allowed revealing the different behaviour of the intact, permeabilised, and lyophilised cells. The following major conclusions can be made on the base of the analysis. The membranes of the intact cells are impermeable for NADH but permeable for different substrates, viz. formaldehyde, methanol, and etha-

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nol. On contrary, the permeabilised cells are permeable for both NAD(P)H and the substrates, but the response current can be seen only after NAD(P)H addition. This fact confirms that the enzyme NADH-ubiquinone oxidoreductase being inside the permeabilised cells is retained in native state but the permeabilised cells have lost mostly all low-molecular weight cofactors. As result of this fact permeabilised cells do not respond to the addition of different substrates. The lyophilised cells proved to be heterogeneous: a part of the cells have the damaged membranes, whereas another part is still native. Indeed, the electrochemical response in the case of lyophilised cells reflects the ratio of the native and damaged cells. Acknowledgements The work was financially supported by INTAS Open Call project No. 03-51-6278 and the Swedish Research Council. References [1] M.J. Allen, Bacteriol. Rev. 30 (1966) 80. [2] F.G. Perez, M. Mascini, I.E. Tothill, A.P.F. Turner, Anal. Chem. 70 (1998) 2380. [3] B.A. Kuznetsov, M.T. Khlupova, S.V. Shleev, A.S. Kaprel’yants, A.I. Yaropolov, Appl. Biochem. Microbiol. 42 (2006) 525. [4] B.A. Kuznetsov, M.E. Davydova, M.O. Shleeva, S.V. Shleev, A.S. Kaprelyants, A.I. Yaropolov, Bioelectrochemistry 64 (2004) 125. [5] P. Ertl, E. Robello, F. Battaglini, S.R. Mikkelsen, Anal. Chem. 72 (2000) 4957. [6] H.N. Li, Y.X. Ci, Anal. Chim. Acta 416 (2000) 221. [7] A. Heiskanen, J. Yakovleva, C. Spegel, R. Taboryski, M. KoudelkaHep, J. Emneus, T. Ruzgas, Electrochem. Commun. 6 (2004) 219. [8] S. Kumar, S.K. Acharya, Anal. Biochem. 268 (1999) 89. [9] K.H.R. Baronian, A.J. Downard, R.K. Lowen, N. Pasco, Appl. Microbiol. Biotechnol. 60 (2002) 108. [10] S. Kerscher, S. Drose, K. Zwicker, V. Zickermann, U. Brandt, Biochim. Biophys. Acta 1555 (2002) 83. [11] D. Pompon, J.-C. Gautier, A. Perret, G. Truan, P. Urban, J. Hepatol. 26 (1997) 81. [12] A.A. Sibirny, G.P. Ksheminskaya. U.S.S.R Patent, Lvovskoe Otdel I Biokhimii Im.A.V.Palladina, USSR, 1991. [13] M.V. Gonchar, G.P. Ksheminska, N.M. Hladarevska, A.A. Sibirny, in: T.M. Lachowicz (Ed.), Genetics of Respiratory Enzymes in Yeasts, Wroclaw University Press, Wroclaw, Poland, 1980, p. 222. [14] M.V. Gonchar, L.B. Kostryk, A.A. Sibirny, Appl. Microbiol. Biotechnol. 48 (1997) 454. [15] M.V. Gonchar, M.M. Maidan, O.M. Moroz, J.R. Woodward, A.A. Sibirny, Biosens. Bioelectron. 13 (1998) 945. [16] M. Khlupova, B. Kuznetsov, O.M. Demkiv, M. Gonchar, E. Cso¨regi, S. Shleev, Talanta 71 (2007) 934. [17] T. Matsunaga, I. Karube, S. Suzuki, Eur. J. Appl. Microbiol. Biotechnol. 10 (1980) 235. [18] D.J. Richardson, Microbiology (UK) 146 (2000) 551. [19] G. Reguera, K.D. McCarthy, T. Mehta, J.S. Nicoll, M.T. Tuominen, D.R. Lovley, Nature 435 (2005) 1098. [20] K. Stott, K. Saito, D.J. Thiele, V. Massey, J. Biol. Chem. 268 (1993) 6097. [21] I. Velazquez, J.P. Pardo, Arch. Biochem. Biophys. 389 (2001) 7. [22] T. Egli, J.P. Van Dijken, M. Veenhuis, W. Harder, A. Fiechter, Arch. Microbiol. 124 (1980) 115.