Oscillation of Membrane Potential in Immobilized DNA Membranes

Sep 29, 2000 - Department of Industrial Chemistry, Seikei University, 3-1 Kichijoji ... Department of Applied Chemistry, Science University of Tokyo, ...
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J. Phys. Chem. B 2000, 104, 9864-9872

Oscillation of Membrane Potential in Immobilized DNA Membranes Akon Higuchi,* Shinya Adachi, Takeshi Imizu, Yoon Boo Ok, Taro Tsubomura, and Mariko Hara Department of Industrial Chemistry, Seikei UniVersity, 3-1 Kichijoji Kitamachi 3, Musashino, Tokyo 180-8633, Japan

Ken Sakai Department of Applied Chemistry, Science UniVersity of Tokyo, 1-3 Kagurazaka, Shinjyuku, Tokyo 162-8601, Japan ReceiVed: April 26, 2000; In Final Form: July 18, 2000

Immobilized DNA membranes prepared by binding between DNA and poly(γ-methyl-L-glutamate) membranes having a cis-diaminedichloro platinum group-generated oscillation of the membrane potential under a concentration gradient of several salts at frequencies between 0 and 5 Hertz at pH 7.0, although nonmodified poly(γ-methyl-L-glutamate) membranes showed a constant potential. The amplitude of oscillation depended on the length of the joint segments between the DNA and poly(γ-methyl-L-glutamate) membranes; the immobilized DNA membranes modified with diaminopropane and diaminobutane showed a high oscillation amplitude (e.g., 4-5 mV). It was also influenced by the salts used, and the highest oscillation amplitude in the membrane potential across the immobilized DNA membranes modified with diaminobutane was observed when NaCl was used as the salt solution. Fast Fourier transport analysis revealed that the oscillation of the membrane potential had several specific frequencies depending on the salt solution and the immobilized DNA membranes used. Recognition of the model endocrine disruptors (i.e., dibenzo-p-dioxin and biphenyl) from the oscillation of the membrane potential was performed in the immobilized DNA membranes. The drastic decrease in the membrane potential and the decrease in magnitude of the power spectra were observed in the immobilized DNA membranes modified with hexamethylenediamine after the immobilized DNA membranes were immersed in the saturated solution of dibenzo-p-dioxin. It was qualitatively possible to recognize intercalating molecules of DNA including the endocrine disruptors from the membrane potential oscillation data of the immobilized DNA membranes.

Introduction Bio-oscillation, that is, pulsating and oscillatory phenomena in cells and living membranes,1-4 is of vital importance in biophysics and physical chemistry. Receptor stimulation by several agonists5 or the epidermal growth factor6 generates a pulsating release of calcium ion from intracellular stores.7 The fluctuations in the intracellular calcium ion concentration cause periodic activation of ion channels,3,8 and spontaneous oscillation in the membrane potential originating from the fluctuation of the calcium ion concentration has been generally observed.9,10 In artificial systems, oscillatory phenomena accompanying a rhythmic change in the redox potential has been studied in solution and gels. The Belousov-Zhabotinsky reaction11-13 is widely known in such phenomena and was explained by the Field-Koros-Noyas mechanism.14 Several kinds of artificial membranes15-24 which induce the oscillation of the membrane potential have also been reported as simple models of cells and living membranes. Most of the studied artificial membranes15-19 are bimolecular lipid or liquid membranes. Only a few studies deal with the electrical oscillatory phenomena in artificial polymeric membranes.20-24 Huang and Spangler21 studied a poly(glutamic acid)-Ca2+ membrane in order to clarify the dynamic behavior of the electrical oscillation. * Author to whom correspondence should be addressed. Fax: +81-42237-3871. E-mail: [email protected].

Minoura et al.22 observed the spontaneous oscillation of the electrical membrane potential in a triblock copolypeptide membrane separating KCl solutions of different concentrations. In our previous study,23 we found that chemically modified poly(γ-methyl-L-glutamate) membranes having diamine segments of several different lengths generated oscillations in the membrane potential under a concentration gradient of several salts at pH ) 10.0. The oscillation of the membrane potential was considered to be caused by the fluctuation in the fixed charge density originating from the thermal fluctuation of the partially ionized amine segments in the modified membranes. The oscillation of the membrane potential in ion-complex membranes of DNA and polyamino acid was also investigated in our previous study.24 Although the amount of DNA immobilized on the polyamino acid membranes was estimated to be small amount, about 1.15 µg/cm2, due to the ion-complex method of the DNA immobilization, it was possible that a particular salt present in the solution can be identified from the amplitude and/or the power spectrum of the fast Fourier transport during the oscillation of the membrane potential.24 Cis-Diaminedichloro platinum(II), cisplatin, is known to bind DNA by 1,2-GG or AG and 1,3-GNG intrastrand cross-links and exhibits an antitumor activity.25-27 Therefore, the immobilized DNA membranes were prepared by binding between DNA and poly(R-amino acid) membranes having a cis-diaminedichloro platinum group in this study. The recognition of

10.1021/jp001600n CCC: $19.00 © 2000 American Chemical Society Published on Web 09/29/2000

Membrane Potential Oscillation in DNA Membranes SCHEME 1

J. Phys. Chem. B, Vol. 104, No. 42, 2000 9865 TABLE 1: Observed Membrane Potential, Calculated Cx/K, and Amount of DNA Immobilized on the Membranes membrane

∆φa (mV)

Cx/K (mol/dm3)

DNA-Pt-EDA-PMLG -108.0 ( 1.0 0.085 ( 0.010 DNA-Pt-DAP-PMLG -102.5 ( 2.5 0.0055 ( 0.001 Pt-DAB-PMLG -73.0 ( 1.0 0.010 ( 0.001 DNA-Pt-DAB-PMLG -65.0 ( 3.0 0.0065 ( 0.001 DNA-Pt-HMD-PMLG 36.5 ( 0.5 -0.0085 ( 0.0005

DNA amount (µg/cm2) 6.3 ( 1.0 6.5 ( 0.9 0 11.7 ( 1.1 5.2 ( 0.4

a Measured at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C.

salts and model chemicals of endocrine disruptors was investigated on the basis of the oscillation of the membrane potential and its power spectra from the fast Fourier transport analysis in the immobilized DNA membranes. Experimental Section Membrane Preparation. Poly(γ-methyl-L-glutamate), PMLG, membranes were prepared by casting a 1 wt % PMLG solution onto flat Petri dishes and then drying at room temperature for 6 days. The membranes having a thickness of 30-35 µm were selected for use in the chemically modified reaction, and the aminolysis reaction of the PMLG membranes (i.e., Scheme 1) was performed by dipping the membranes in ethylenediamine (EDA), 1,3-diaminopropane (DAP), 1,4-diaminobutane (DAB), and hexamethylenediamine (HMD) at 25 °C (for the EDA and DAP reactions) or 50 °C (for the DAB and HMD reactions) for 6 h.23 The modified membranes were rinsed with ethanol and subsequently dried under vacuum at room temperature. The diaminated ratio was estimated from an atomic analysis of the membranes, and the diaminated ratio under the described conditions was estimated to be 0.21 ( 0.02 for the EDA modification (EDA-PMLG), 0.12 ( 0.01 for the DAP modification (DAP-PMLG), 0.24 ( 0.02 for the DAB modification (DAB-PMLG), and 0.15 ( 0.02 for the HMD modification (HMD-PMLG) in this study (see Scheme 1). An amount of 1mmol of K2[PtCl4] was dissolved in 25 mL of ultrapure water at 40 °C. The diaminated PMLG (EDAPMLG, DAP-PMLG, DAB-PMLG, and HMD-PMLG) membranes were immersed in the K2[PtCl4] solution at 25 °C for 8 h to prepare the cis-[PtCl2(diaminated PMLG)] membranes. The modified membranes were rinsed with ultrapure water and immersed in ultrapure water for 24 h. The degree of platinum binding was estimated from an atomic analysis of the membranes, and the degree of platinum binding under these conditions was estimated to be 2.1 ( 0.2 µmol/cm2 for Pt modification of the EDA-PMLG (Pt-EDA-PMLG) membranes, 1.9 ( 0.2 µmol/cm2 for Pt modification of the DAP-PMLG (PtDAP-PMLG) membranes, 4.3 ( 0.5 µmol/cm2 for Pt modification of the DAB-PMLG (Pt-DAB-PMLG) membranes, and 1.7 ( 0.2 µmol/cm2 for Pt modification of the HMD-PMLG (PtHMD-PMLG) membranes in this study (see Scheme 1). DNA from salmon testes (D-1626, Sigma Chem. Co.) was dissolved in a buffer containing 10 mM Tris-HCl and 1.0 mM EDTA. After the DNA solution was adjusted to a pH of 7.0 by adding 0.1 M acetic acid and diluted to a concentration of 500 µg/mL by adding the buffer, cis-[PtCl2(diaminated PMLG)]

membranes were dipped into the DNA solution for 12 h and the immobilized DNA membranes where DNA was bound to the cis-[PtCl2(diaminated PMLG)] membranes (i.e., DNA-PtEDA-PMLG, DNA-Pt-DAP-PMLG, DNA-Pt-DAB-PMLG, and DNA-Pt-HMD-PMLG membranes) were prepared using these procedures (see Scheme 1). The amount of DNA immobilized on the cis-[PtCl2(diaminated PMLG)] membranes was estimated to be 6.3 ( 1.0 µg/cm2 for the DNA-Pt-EDA-PMLG membranes, 6.5 ( 0.9 µg/cm2 for the DNA-Pt-DAP-PMLG membranes, 11.7 ( 1.1 µg/cm2 for the DNA-Pt-DAB-PMLG membranes, and 5.2 ( 0.4 µg/cm2 for the DNA-Pt-HMD-PMLG membranes on the basis of an atomic analysis of phosphate atom in the immobilized DNA membranes (see Table 1). Membrane Potential Measurements. The membrane potential, ∆φ, was measured in cells that consisted of two chambers separated by the membranes.28-31 The concentrations of the aqueous salt solutions were 1 mM in one side of the chamber (side 1), C1, and 0.1 M in the other side of the chamber (side 0), Co. The revolution speed of the magnetic spinbars in the cells was controlled (i.e., 230 rpm in this study) by magnetic stirrers. The potential was measured using a digital multimeter (model 7561, Yokogawa Electronic Co.) using Ag/AgCl electrodes (TOA HS-205C, TOA Electronics, Ltd.), and the data were transferred to a 32-bit personal computer (PC-9801BX, NEC Corp).29-31 The pH in the cell was monitored with a pH meter (TOA HM-30S, TOA Electronics, Ltd.) and was adjusted to pH 7.0 by introduction of a 0.01 M NaOH solution into the cells. The solution in the cell was replaced with ultrapure water several times after each set of measurements. Each of the membranes was capable of withstanding more than 300 measurements over a three-month period. Figures 2-11 show typical curves which were selected from forty measurements using four different membranes that were performed under the same conditions (1200 data sampling points for 120 s at one experiment). Results and Discussion Characterization of the DNA Membranes. The EPMA spectra on the surface of the DNA-Pt-HMD-PMLG membranes were investigated and are shown in Figure 1. In the figure, the concentration of Pt from diaminodichloro platinum and the concentration of P from DNA on the membranes were observed to be higher than that on the background and remained constant at all points on the DNA-Pt-HMD-PMLG membranes. Oscillation of Membrane Potential. The time course of the membrane potential was investigated in the PMLG, DABPMLG, Pt-DAB-PMLG, and DNA-Pt-DAB-PMLG membranes at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C and is shown in Figure 2. Nonmodified PMLG and DAB-PMLG membranes showed a constant potential within 0.3 mV variation and can be considered to show no oscillation at pH 7.0. These results are consistent based on our previous study where no oscillation was observed in PMLG and diaminated PMLG membranes at pH < 8.0.23

9866 J. Phys. Chem. B, Vol. 104, No. 42, 2000

Higuchi et al.

Figure 2. Time course of membrane potential across PMLG (a), DABPMLG (b), Pt-DAB-PMLG (c), and DNA-Pt-DAB-PMLG (d) membranes between 1.0 mM NaCl solution and 0.1 M NaCl solution at pH ) 7.0 and 37 °C. The data shown are representative of forty experiments using four different membranes.

Figure 1. EPMA(EDX) spectra of Pt (a) and P (b) for the surface of DNA-Pt-HMD-PMLG membrane.

The membrane potential was, on the other hand, observed to generate spontaneous oscillation in both the Pt-DAB-PMLG and DNA-Pt-DAB-PMLG membranes. The amplitude of the oscillation in the DNA-Pt-DAB-PMLG membranes was found to be more than 5 mV and was greater than that in the Pt-DABPMLG membranes. In our previous study,24 oscillation of the membrane potential was also observed in ion-complex membranes of DNA and HMD, of which the immobilized DNA was only 1.15 µg/cm2 compared to 5.2-11.7 µg/cm2 in the immobilized DNA membranes in this study (see Table 1). The membrane potential in a homogeneous charged membranes measured with a univalent cation and a univalent anion is expressed by the Teorell-Meyer-Sievers (TMS) theory.32-37

∆φ ) -

{

}

[1 + 4y12]1/2 - RU C1[1 + 4yo2]1/2 - R RT + U ln ln zF Co[1 + 4y12]1/2 - R [1 + 4yo2]1/2 - RU (1)

where U ) [ξ+ - ξ-]/[ξ+ + ξ-], ξ+, and ξ- are the mobilities

of the cation and the anion, yo ) KCo/Cx, y1 ) KC1/Cx, Cx is the effective fixed charge concentration,32-36 K is the thermodynamic partition coefficient,32-36 R has a value of +1 or -1 when the membrane is positively or negatively charged, respectively, z is the valence of the ion (z ) 1 in this study), and R, T, and F have their conventional meanings. The only unknown parameters in eq 1 are Cx/K and U. Cx/K is known to have a predominant influence on the membrane potential.32 Cx/K was estimated from the value of the membrane potential in the Pt-DAB-PMLG and DNA-Pt-DAB-PMLG membranes between 1.0 mM and 0.1 M NaCl using eq 1 and is summarized in Table 1. The mobilities of the sodium and chloride ions used in the calculations were selected to be those in bulk water, 5.382 × 10-13 m2 mol s-1 J-1 and 8.201 × 10-13 m2 mol s-1 J-1, respectively.38 Cx/K of DNA-Pt-DAB-PMLG membranes was found to be 0.0035 mol/dm3 less than that of Pt-DAB-PMLG membranes (see Table 1). Although the immobilized DNA membranes prepared in this study have positively fixed-charged sites (diamine groups) and negatively fixed-charged sites (DNA), DNA conjugated on the surface of the DNA-Pt-DAB-PMLG membranes contributed to more negatively fixed-charged density of 0.0035 mol/dm3 than that on the Pt-DAB-PMLG membranes. The effect of joint segments (i.e., diamines such as EDA, DAP, DAB, and HMD) between Pt and PMLG on the oscillation of the membrane potential was investigated. Figure 3 shows time course of the membrane potential in DNA-Pt-EDA-PMLG, DNA-Pt-DAP-PMLG, DNA-Pt-DAB-PMLG, and DNA-PtHMD-PMLG membranes at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C. The membrane potential of DNAPt-EDA-PMLG was found to be a negative value around -108 mV, while the membrane potential of DNA-Pt-HMD-PMLG was a positive value around 37 mV. The value of the membrane potential increased from approximately -108 mV to 37 mV

Membrane Potential Oscillation in DNA Membranes

Figure 3. Time course of membrane potential across DNA-Pt-EDAPMLG (a), DNA-Pt-DAP-PMLG (b), DNA-Pt-DAB-PMLG (c), and DNA-Pt-HMD-PMLG (d) membranes between 1.0 mM NaCl solution and 0.1 M NaCl solution at pH ) 7.0 and 37 °C. The data shown are representative of forty experiments using four different membranes.

with the increase in the length of diamine (e.g., from EDA to HMD) in the immobilized DNA membranes. Cx/K was also estimated from the values of the membrane potential in the DNA-Pt-EDA-PMLG, DNA-Pt-DAP-PMLG, DNA-Pt-DAB-PMLG, and DNA-Pt-HMD-PMLG membranes using eq 1 and is summarized in Table 1. DNA-Pt-EDA-PMLG, DNA-Pt-DAP-PMLG, and DNA-Pt-DAB-PMLG membranes were estimated to be positively charged membranes (Cx/K ) 0.0055-0.085 mol/dm3) where the fixed charge is predominantly contributed by the diamine groups rather than the phosphate group of DNA in the immobilized DNA membranes. Only the DNA-Pt-HMD-PMLG membranes showed the membrane potential of negatively charged membranes, and Cx/K in the membranes was estimated to be -0.0085 mol/dm3 (see Table 1). This indicates that DNA on the DNA-Pt-HMD-PMLG membranes contributes to a higher amount of negatively fixedcharge density in the membranes compared to DNA on the other immobilized DNA membranes in this study. Because the amount of DNA conjugated on the DNA-Pt-HMD-PMLG membranes is almost the same or relatively smaller than that on the other immobilized DNA membranes (see Table 1), DNA on the DNAP-HMD-PMLG membranes is expected to become attached in more condensed and compacted form compared to DNA on the other immobilized DNA membranes, which enhances the negatively fixed-charge density in the membranes. The amplitude of the oscillation in the membrane potential was observed to increase in the following order at Co ) 0.1 M, C1 ) 1 mM, pH 7.0, and 25 °C: DNA-Pt-HMD-PMLG ) DNA-Pt-EDA-PMLG < DNA-Pt-DAP-PMLG ) DNA-PtDAB-PMLG (e.g., the amplitude of the oscillation in the DNAPt-HMD-PMLG membranes was statistically different from that of the DNA-Pt-DAP membranes or DNA-Pt-DAB-PMLG membranes (p < 0.03), but was not statistically different from

J. Phys. Chem. B, Vol. 104, No. 42, 2000 9867 that of the DNA-Pt-EDA-PMLG membranes (p > 0.1). The amplitude of the oscillation in the DNA-Pt-DAP-PMLG membranes was not statistically different from that in the DNA-PtDAB-PMLG membranes (p > 0.1).). This result indicates that an adequate length of joint segments between the DNA and PMLG is necessary for enhanced oscillation of the membrane potential. The joint segment of EDA is probably so short that DNA on DNA-Pt-EDA-PMLG might find it difficult to freely fluctuate. On the other hand, DNA on DNA-Pt-HMD-PMLG might be so condensed on the membrane surface that the fluctuation of the phosphate group in DNA is rather difficult and might be more restricted than DNA on the other immobilized DNA membranes. Models of the Oscillation. Oscillation of the membrane potential in some artificial polymeric membranes was reported under a constant gradient of salt solution or a dc electric field.20-22 The oscillation of membrane potential was reported to show spikes or periodic pulses and was unlike our oscillatory features such as those shown in Figures 2-4, 6, 7, 9, and 10. Their models for the oscillation phenomena were based on a change in polymeric conformation induced by salt accumulation at the interfacial region in the membranes21 or by a periodic change in the conformation between the R-helix and the random coil structure of the poly(amino acid) chain.22 Their models cannot be applied to the oscillation of membrane potential in the immobilized DNA membranes in this study. The oscillation of membrane potential in the immobilized DNA membranes appears unlike their electrical patterns of spikes and periodic pulses but rather shows several specific peaks based on the analysis of fast Fourier transform. It is, therefore, proposed that the oscillation in membrane potential is caused by the fluctuation of the fixed charge density originating from the thermal fluctuation of the phosphate group of DNA in the immobilized DNA membranes under a constant gradient of salts as is suggested in the previous study.24 Recognition of Salts. One might think that the amplitude or the components of specific frequencies in the oscillation of the membrane potential analyzed by the fast Fourier transport method39 vary when the various salt solutions were used in the measurements, and the recognition of salts would be qualitatively possible based on the amplitude and the components of the specific frequencies. Several salts (i.e., LiCl, NaCl, KCl, CsCl, CaCl2, and MgCl2) were used in the measurements of the oscillation of the membrane potential in the DNA-Pt-DAP-PMLG and DNA-PtHMD-PMLG membranes. Figure 4 shows the oscillation of the membrane potential in the DNA-Pt-DAP-PMLG membranes measured using NaCl, KCl, MgCl2, and CaCl2 solutions at Co ) 0.1 M, C1 ) 1 mM, pH 7.0, and 37 °C. The amplitude of the oscillation of the membrane potential measured using the salt solutions was observed to increase in the following order: MgCl2 < CaCl2 ) KCl < NaCl (e.g., the amplitude of the oscillation measured using CaCl2 was not statistically different from that measured using KCl (p > 0.1), but was statistically different from MgCl2 and NaCl (p < 0.03)). From these results, it is difficult to recognize the species of salts only from the amplitude of the oscillation. Therefore, the oscillation of the membrane potential measured using several salt solutions was analyzed using a fast Fourier transport method39 to investigate whether the power spectra of the oscillation in the membrane potential can recognize the specific salts. Figure 5 shows the power spectra of the oscillation in the membrane potential measured using NaCl, KCl, MgCl2, and CaCl2 solutions in the DNA-Pt-DAP-PMLG membranes. Several specific peaks depending on the salt solutions were observed in the figure. Only a broad peak around 0.1 Hz was observed

9868 J. Phys. Chem. B, Vol. 104, No. 42, 2000

Figure 4. Time course of membrane potential across DNA-Pt-DAPPMLG membranes between 1.0 mM salt solution and 0.1 M salt solution at pH ) 7.0 and 37 °C. The salt solutions used as the driving force of membrane potential were NaCl (a), KCl (b), MgCl2 (c), and CaCl2 (d) solutions. The data shown are representative of forty experiments using four different membranes.

Figure 5. Power spectra of the oscillation in the membrane potential across DNA-Pt-DAP-PMLG membranes measured using salt solution of NaCl, KCl, MgCl2, and CaCl2 at Co ) 0.1 M, C1 ) 1 mM, pH ) 7.0, and 37 °C. The data shown are representative of forty experiments using four different membranes.

in the power spectra of the oscillation measured using the KCl and MgCl2 solutions. The magnitude of the power spectra for KCl solution was found to be higher than that for the MgCl2 solution. Several specific peaks every 0.2-0.3 Hz were observed in the power spectra of the oscillation measured using NaCl and CaCl2 solutions. The magnitude of the power spectra for NaCl was found to be higher than that for the CaCl2 solution. The magnitude of the power spectra for LiCl and CsCl was also found to be more than 0.4 at 0.1 Hz (data not shown). The power spectra for salt solutions containing univalent cations

Higuchi et al.

Figure 6. Time course of membrane potential across DNA-Pt-HMDPMLG membranes between 1.0 mM salt solution and 0.1 M salt solution at pH ) 7.0 and 37 °C. The salt solutions used as the driving force of membrane potential were NaCl (a), KCl (b), MgCl2 (c), and CaCl2 (d) solutions. The data shown are representative of forty experiments using four different membranes.

always showed a higher magnitude than those for salt solutions containing divalent cations in this study. The patterns of frequencies that appeared in the power spectra showed exactly the same tendencies in the power spectra that were measured using the different membranes prepared under the same conditions. These results suggest that it may be possible to recognize a particular salt present in the solution from the information based on the amplitude and the power spectra of fast Fourier transport during the oscillation of membrane potential to some extent. The oscillation of the membrane potential in the DNA-PtHMD-PMLG membranes using NaCl, KCl, MgCl2, and CaCl2 solutions was also investigated at Co ) 0.1 M, C1 ) 1 mM, pH 7.0, and 37 °C and is shown in Figure 6. The sign of the membrane potential measured using the NaCl and KCl solutions was observed to be positive and that measured using the LiCl and CsCl solutions was also found to be positive (data not shown), while the sign of the membrane potential measured using CaCl2 and MgCl2 was found to be the opposite sign, i.e., negative. The explanations for the observed negative sign in the membrane potential using the salt solution containing a divalent cation is that (1) the diffusion potential of CaCl2 and MgCl2 in the membranes is negative because the mobility of cation is lower than the mobility of anion in the solution from the membrane potential theory,37 and (2) the divalent cation was adsorbed on the DNA-Pt-HMD-PMLG membranes and the adsorbed cation on the membranes contributes to the positively charged membranes, which produces the negative sign of the membrane potential at Co ) 0.1 M, C1 ) 1 mM. After the membrane potential of DNA-Pt-HMD-PMLG membranes was measured in CaCl2 solution, the membrane potential was also measured in NaCl solution to test whether

Membrane Potential Oscillation in DNA Membranes

Figure 7. Time course of membrane potential across DNA-Pt-HMDPMLG membranes between 1.0 mM salt solution and 0.1 M salt solution at pH ) 7.0 and 37 °C. The salt solutions used as the driving force of membrane potential were NaCl solution (a, the first measurement), CaCl2 (b, the second measurement), NaCl solution (c, the third measurement), and NaCl solution (d, the fourth measurement). The membrane was washed with 0.2 wt % EDTA solution at pH 7.0 after the measurement of NaCl solution at the third measurement (i.e., (c)). The data shown are representative of forty experiments using four different membranes.

reproducible data could be obtained and the initial membrane potential could be observed using the NaCl solution. Figure 7 shows the time course of the membrane potential in the DNA-Pt-HMD-PMLG membranes where the (a) NaCl solution (the first measurement), (b) CaCl2 solution (the second measurement), (c) NaCl solution (the third measurement), and (d) NaCl solution (the fourth measurement) were used successively at Co ) 0.1 M, C1 ) 1 mM, pH 7.0, and 37 °C. The membrane was washed with 0.2 wt % EDTA solution at pH 7.0 after the measurements of the NaCl solution at the third measurement. The value of the membrane potential using the NaCl solution (c) was reduced to 9 (1 mV (e.g., initial membrane potential is 37 ( 1 mV at (a)) after the measurements of the membrane potential using CaCl2 solution. Therefore, the membrane was washed using the 0.2 wt % EDTA solution for removal of the adsorbed Ca ion with a chelating agent of EDTA, because the fixed charge density was reported to be changed by the ion adsorption on the membranes.40-42 The membrane potential using NaCl solution during the fourth measurement (d) was observed to recover to the initial value of the membrane potential, i.e., 37-39 ( 1 mV. This result indicates that the negative sign of the membrane potential measured using the CaCl2 solution is due to not only the negative value of the diffusion potential of CaCl2 in the membranes but also the adsorption of divalent cations on the DNA-Pt-HMD-PMLG membranes. In this study, the immobilized DNA membranes were washed with 0.2 wt % EDTA solution every time after the measurements of the membrane potential using the salt solution containing the divalent cations.

J. Phys. Chem. B, Vol. 104, No. 42, 2000 9869

Figure 8. Power spectra of the oscillation in the membrane potential across DNA-Pt-HMD-PMLG membranes measured using salt solution of NaCl (a) and CaCl2 (b) at Co ) 0.1 M, C1 ) 1 mM, pH ) 7.0, and 37 °C. The data shown are representative of forty experiments using four different membranes.

The power spectra of the oscillation in the membrane potential were measured using LiCl, NaCl, KCl, CsCl, MgCl2, and CaCl2 solutions in the DNA-Pt-HMD-PMLG membranes for the recognition of salts from their specific frequencies. Figure 8 shows the power spectra measured using the NaCl and CaCl2 solution as examples. The magnitude of the power spectra in the DNA-Pt-HMD-PMLG membranes is significantly less than that observed in the DNA-Pt-DAP-PMLG membranes (see Figure 5). This is due to the lower amplitude of the oscillation in the DNA-Pt-HMD-PMLG membranes than that in the DNAPt-DAP-PMLG membranes. The specific peak at 4 Hz was always observed in the DNA-Pt-HMD-PMLG membranes in the power spectra measured using any of the salt solutions in this study. The patterns of the specific frequencies observed in the power spectra were almost the same among the salts for both the univalent and divalent cations used in this study. Therefore, it is difficult to recognize the specific salts in the solution from the power spectra of the oscillation in the membrane potential of the DNA-Pt-HMD-PMLG membranes, although the univalent and divalent cations in the salt solution can be recognized from the sign of the membrane potential. Recognition of Model Endocrine Disruptors. DNA is known to show specific binding with several antibiotic and antitumor drugs which intercalatively bind to the double helical DNA. The antitumor action of the drugs that covalently bind to DNA is believed to improve the selective killing of tumor cells.43 The chemicals having aromatic rings and hydrophobic characteristics such as fluorescent dyes of Pico Green,44 Hoechest 33258,45 and acridine orange46 can also intercalatively bind to DNA between bases. Kato et al. reported the removal of model endocrine disruptors with the absorbent of insolubilized DNA by the formation of an intercalating and stacking complex of the endocrine disruptors with DNA.47

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Figure 9. Time course of membrane potential across DNA-Pt-DABPMLG membranes treated before (a) and after the saturated solution of dibenzo-p-dioxin (b) and power spectra (c) of the oscillation in the membrane potential across DNA-Pt-DAB-PMLG membranes treated before (broken line) and after the saturated solution of dibenzo-p-dioxin (solid line) at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C. The data shown are representative of forty experiments using four different membranes.

Therefore, recognition of the model endocrine disruptors was investigated from the oscillation of the membrane potential and its power spectra of the fast Fourier transport analysis in the DNA-Pt-DAB-PMLG and DNA-Pt-HMD-PMLG membranes which were treated with and without the saturated solution of the endocrine disruptors (i.e., dibenzo-p-dioxin and biphenyl as a model of PCB) in this section. Figure 9 shows the time course of the membrane potential in the DNA-Pt-DAB-PMLG membranes treated with and without the saturated solution of dibenzo-p-dioxin (4.0 ppm) at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH 7.0, and 37 °C. No apparent difference in the membrane potential was observed before and after the treatment of the saturated dibenzo-p-dioxin, although an increase of 5 mV observed in the membrane potential was not statistically relevant (p > 0.03) after the DNAPt-DAB-PMLG membranes were immersed in the saturated dibenzo-p-dioxin solution. The power spectra of the oscillation in the membrane potential in the DNA-Pt-DAB-PMLG membranes treated with and without the saturated dibenzo-p-dioxin solution were also investigated and are shown in Figure 9c. Several small but specific peaks were observed at every 0.8-1.2 Hz in the power spectra of the membrane potential in the DNA-Pt-DAB-PMLG membranes before the treatment with dibenzo-p-dioxin, while almost no peak was observed in the DNA-Pt-DAB-PMLG membranes after the treatment with dibenzo-p-dioxin. No significant change in the oscillation of membrane potential and its power spectra was observed before and after the treatment

Higuchi et al.

Figure 10. Scheme of dibenzo-p-dioxin and biphenyl (a) and time course of membrane potential across DNA-Pt-HMD-PMLG membranes treated before (b) and after the saturated solution of biphenyl (c) and dibenzo-p-dioxin (d) at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C. The data shown are representative of forty experiments using four different membranes.

using saturated dibenzo-p-dioxin solution in the DNA-Pt-DABPMLG membranes. Recognition of dibenzo-p-dioxin and biphenyl from the oscillation of the membrane potential was also performed in the DNA-Pt-HMD-PMLG membranes which showed a positive sign for the membrane potential at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH 7.0, and 37 °C. Figure 10 shows the time course of the membrane potential in the DNA-Pt-HMD-PMLG membranes treated with and without the saturated solution of dibenzo-p-dioxin (4.0 ppm) and biphenyl (7.5 ppm). The amplitude of the oscillation of the membrane potential in the DNA-Pt-HMD-PMLG membranes was found to increase when the membranes were treated with the saturated solution of dibenzo-p-dioxin (approximately 2 mV amplitude) and biphenyl (approximately 3-4 mV amplitude), while the amplitude of the oscillation was only 1 mV in the DNA-Pt-HMD-PMLG membranes before the treatment of the model endocrine disruptors. A drastic decrease in the membrane potential was observed when the DNA-Pt-HMD-PMLG membranes were treated with the saturated solution of dibenzo-p-dioxin (e.g., from 37 ( 1 mV to 23 ( 2 mV) and biphenyl (e.g., from 37 ( 1 mV to -15 ( 3 mV). The membrane potential in the DNA-Pt-HMDPMLG membranes shifted from a positive sign before the treatment to a negative sign after treatment with the biphenyl solution. It is widely known that the conformational flexibility of the DNA backbone is reduced when the drug molecules intercalate with DNA.48 Therefore, DNA layers on the surface of the DNAPt-HMD-PMLG membranes may be swollen in the aggregated DNA layers when the DNA backbones have less flexibility due to the intercalation of the dibenzo-p-dioxin and biphenyl. One of the explanations for the decrease in the membrane potential

Membrane Potential Oscillation in DNA Membranes

Figure 11. Power spectra of the oscillation in the membrane potential across DNA-Pt-HMD-PMLG membranes treated before (a) and after the saturated solution of dibenzo-p-dioxin (b) and biphenyl (c) at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C. The data shown are representative of forty experiments using four different membranes.

is that the DNA layer on the DNA-Pt-HMD-PMLG membranes reduces the compactness and rigidness due to the intercalation of the dibenzo-p-dioxin and biphenyl. The contribution of the negative charge density from the phosphate group may subsequently decrease and the contribution of the amine of PMLG may increase in the DNA-Pt-HMD-PMLG membranes, because the negative sign of the membrane potential at Co ) 0.1 M, C1 ) 1 mM, and pH 7.0 indicates that the membranes are positively charged due to the amine group in PMLG and the positive sign of the membrane potential at Co ) 0.1 M, C1 ) 1 mM, and pH 7.0 indicates that the membranes are negatively charged due to the phosphate group of DNA in the DNA-Pt-HMD-PMLG membranes on the basis of the membrane potential theory of charged membranes31-34 (i.e., eq 1). The power spectra of the oscillation in the membrane potential in the DNA-Pt-HMD-PMLG membranes treated with and without the saturated solution of dibenzo-p-dioxin and biphenyl were investigated, and are shown in Figure 11. The specific peaks at 4 Hz and around 0.1-0.5 Hz were found in all cases as shown in the figure. The magnitude of the power spectra at 4 Hz decreased when the DNA-Pt-HMD-PMLG membranes were immersed in the saturated solution of dibenzo-p-dioxin, while the magnitude of the power spectra at 4 Hz increased for the DNA-Pt-HMD-PMLG membranes treated with the saturated solution of biphenyl. A new peak at 0.3 Hz appeared in the power spectra of the oscillation in the DNA-Pt-HMD-PMLG membranes treated with the saturated solution of biphenyl. The magnitude of the power spectra varied (50% on average at specific frequencies depending on the time measured and membranes prepared in this study. The probabilities of the magnitude of the power spectra at 4 Hz were, therefore, investigated using 10 experiments on every four membranes (i.e., 40 total experiments; 1200 membrane potential points for 120 s in one experiment), and are shown in Figure 12. These results also indicate that the magnitude of the power spectra at 4 Hz decreased for the DNA-Pt-HMD-PMLG membranes treated with the saturated solution of dibenzo-p-dioxin and that the magnitude of the power spectra at 4 Hz increased for the DNA-Pt-HMD-PMLG membranes treated with the saturated solution of biphenyl.

J. Phys. Chem. B, Vol. 104, No. 42, 2000 9871

Figure 12. The probabilities of the magnitude of power spectra at 4 Hz in the oscillation of membrane potential across DNA-P-HMDPMLG membranes treated before (O) and after the saturated solution of dibenzo-p-dioxin (9) and biphenyl (b) at Co ) 0.1 M NaCl, C1 ) 1 mM NaCl, pH ) 7.0, and 37 °C. The data were calculated from forty experiments using four different membranes.

The decrease or increase in the magnitude of the power spectra depending on the intercalating molecules is explained by the change in the conformational flexibility of DNA depending on the intercalating molecules and/or the difference in binding constants of the intercalating molecules to DNA in the DNA-Pt-HMD-PMLG membranes. These results suggest that specific intercalating molecules including the endocrine disruptors will be recognized on the basis of the amplitude and power spectra of fast Fourier transform information during the oscillation of the membrane potential in the DNA-Pt-HMD-PMLG membranes treated with and without the solution of intercalating molecules. Concluding Remarks. The immobilized DNA membranes prepared from binding between DNA and poly(γ-methyl-Lglutamate) membranes having a cis-diaminedichloro platinum group had a higher amount of immobilized DNA (i.e., 5.2-1.7 µg/cm2) on the membranes compared to the DNA membranes prepared from the ion-complex of DNA and polyamino acid in our previous study.24 The oscillation of the membrane potential across the immobilized DNA membranes prepared in this study was observed under a concentration gradient of several salts at frequencies between 0 and 5 Hertz at pH 7.0, while nonmodified poly(γ-methyl-L-glutamate) membranes showed a constant potential. The oscillation in the membrane potential was considered to be caused by the fluctuation in the fixed charge density originating from the thermal fluctuation of the phosphate group of DNA in the DNA membranes under a constant gradient of salt solution. The amplitude of the oscillation depended on the length of the joint segments between the DNA and poly(γ-methyl-Lglutamate) membranes, and the immobilized DNA membranes modified with diaminopropane and diaminobutane showed a high oscillation amplitude (e.g., 4-5 mV). It was also influenced by the salts used, and the highest oscillation in the membrane potential across the immobilized DNA membranes modified with diaminobutane was observed when NaCl was used as the salt solution. Fast Fourier transport analysis revealed that the oscillation of the membrane potential had several specific frequencies depending on the salt solution and the immobilized DNA membranes used. Recognition of model endocrine disruptors (i.e., dibenzo-pdioxin and biphenyl) from the oscillation of the membrane

9872 J. Phys. Chem. B, Vol. 104, No. 42, 2000 potential was performed in the immobilized DNA membranes. The drastic decrease in the membrane potential and the decrease in the magnitude of the power spectra were observed in the immobilized DNA membranes modified with hexamethylenediamine, after the immobilized DNA membranes were immersed in the saturated solution of dibenzo-p-dioxin. It was qualitatively possible to recognize the intercalating molecules of DNA including the endocrine disruptors from the oscillation results of the membrane potential in the immobilized DNA membranes. The olfactory and taste systems in animals discriminate and recognize thousands of olfactory and taste compounds. One theory of the enormous capacity of chemical recognition is based on the existence of a large family of olfactory and taste receptors comprised of several thousand subtypes.49,50 Another theory is based on the nonlinear processing of signal information generated by limited types of receptors combined with thousands of guest compounds (i.e., olfactory and taste compounds). In this case, the signal information is processed to recognize the thousands of compounds on the basis of signal amplitude, interval, decay, and oscillation frequency.51 Therefore, the present membrane potential oscillation in the immobilized DNA membranes varied with the salt used or with the treatment of the model endocrine disruptors will present a simple processing mode of recognition for smell and taste in animal brains.24 References and Notes (1) Ritter, M.; Woll, E.; Waldegger, S.; Haussinger, D.; Lang, H. J.; Scholz, W.; Scholkens, B.; Lang, F. Pflugus Arch. 1993, 423, 221-224. (2) Lang, F.; Friedrich, F.; Kahn, E.; Woll, E.; Hammerer, M.; Waldegger, S.; Maly, K.; Grunicke, H. J. Biol. Chem. 1991, 266, 49384942. (3) Stelling, J. W.; Jacob, T. J. C. Am. J. Physiol. 1993, 265, C720C727. (4) Pickering, A. E.; Spanswick, D.; Logan, S. D. J. Physiol. 1994, 480, 109-121. (5) Devor, D. C.; Simasko, S. M.; Duffey, M. E. Am. J. Physiol. 1991, 260, C598. (6) Enomoto, K.-I.; Cossu, M. F.; Edwards, C.; Oka, T. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4754. (7) Crawford, K. M.; Stuenkel, E. L.; Ernst, S. A. Am. J. Physiol. 1991, 261, C177-C184. (8) Devor, D. C.; Simasko, S. M.; Duffey, M. E. Am. J. Physiol. 1990, 258, C318-C326. (9) Ince, C.; Leijh, P. C. J.; Meijer, J.; Bavel, E. van; Ypey, D. L. J. Physiol. (London) 1984, 352, 625-635. (10) Okada, Y.; Tsucjiya, W.; Yada, T. J. Physiol. (London) 1982, 327, 449-461. (11) Zaikin, A. N.; Zhabotinsky, A. M. Nature 1970, 225, 535-537. (12) Reusser, E. J.; Field, R. J. J. Am. Chem. Soc. 1979, 101, 10631071. (13) Yoshida, R.; Onodera, S.; Yamaguchi, T.; Kokufuta, E. J. Phys. Chem. A 1999, 103, 8573-8578. (14) Field, R. J.; Koros, E.; Noyes, R. M. J. Am. Chem. Soc. 1972, 94, 8649-8664. (15) Teorell, T. J. Gen. Physiol. 1959, 42, 831-845.

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