Electrochemical studies of Meldola blue-modified bilayer lipid membranes DING Lin and WANG Erkang * Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022. China Keywords:
Meldola blw, bilayer llpid membranes (BI.M), electrochemistry.
BILAYER lipid membranes (B1.M) have been extensively regarded as a basic structure that forms the matrix of biological membranes"'. The properties of BLM may be altered by incorporation and modification and can be employed to study many membrane processesr2.3 1 . Especially biosensors and bi-molecular electronic devices made by BLM have a wide application[41. Many kinds of organic dyes are known as biological stains and often used in biology, physiology, pharmacology and medicine. In general, coloring and staining processes involve adsorption, solubility, penetration, and chemical reaction, but how dyes affect lipid membranes is still unknown[s1. In this work, a biocatalyst, Meldola blue which is used to prepare biosensors is chosen to modify BLM and its transferring properties are also analyzed.
1 Experimental 1 . 1 Reagents and preparation of solutions Major reagents: natural egg lecithin (NPC) (East China Normal University Chemicals, Shanghai) was chemical-pure, L-a-phosphatidylglycerol (PG) (Sigma Co.,' USA) and Meldola blue ( MB) ( Schmid GmbH, Germany) were all analytically pure. Oxidized cholesterol (OCH) was prepared according to ref. [ 11. Decane and butanol (No. 1 Reagent Factory, Shanghai) were chemical-pure and analytically pure respectively. The former was first purified by distillation and then the two reagents were filtrated through an active alumina column. Superpurified water was obtained from MiIIi-QII (Millipore Co, USA). Other chemicals were analytically pure. The concentration of lipids in the membrane-forming solution was 20 mg/mL in the mixture of decane and butanol with volume ratio 1: 1. The supporting electrolyte was 0 . 1 mol/L NaC1. The buffer was B. R . solution with different pH values. 1 . 2 Measuring instruments and apparatus The electrochemical measurement system was performed by using a CS-1087 computer-
*
T o whom correspondence shonld be addressed.
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controlled electroanalytical system ( Cypress Systems, Inc., USA, donated by Prof. Kuwana, University of Kansas ) . The computer was 386 PC/AT ( Casper Co, Taiwan). The syringe was Hamilton (Hamilton Co, USA). As shown in fig. 1, the cell was a sandwich type, two-compartment one. Two Lucite compartments with almost the same volume were fixed together by several screws, and a Teflon film ( 0 . 1 mm thickness) with a 1-mm aperture U/ was fixed between the two Lucite compartments. Fig. 1. Schematic structure of sandwich-type two-compartment A two-electrode system, two saturatcell. 1, Cell body; 2, aperture for forming BLM; 3, stirring bar; ed calomel electrodes were put into the two 4, fixing screw; 5, Teflon film. compartments respectively, which, used by Tien et a t . [ 6 1 , were employed to fulfill the electrochemical measurement.
1 . 3 Preparation of BLM The preparation of BLM is as described in ref. [ 7 ] . 5 pL membrane-forming solution was injected into the aperture with a Hamilton syringe. After the membrane was formed (about 10 min) , the electrochemical measurement .was carried out.
2 Results and discussion 2 . 1 Influence of membrane-forming lipids on the transport of MB across BLM Three different lipids are used to form different BLMs: NPC, NPC + OCH (the mole fraction of NPC and OCH is 2: 1 ) and PG (the concentration of total lipids in the membraneforming solution in all situations is 20 mg/mL) . Fig. 2 is the typical cyclic voltammograms of MB transport across BLM. It clearly shows that the voltammetric characteristics of BLM are obviously changed after 1 0 - ~ m o l /MB ~ is added to the cis-side of the membrane (the side of the working electrode) and the basic characteristics of dye-modified BLM also appear. Fig. 2 shows that the current is outstandingly increased after MB is added to one side of the membrane. This result indicates that MB is adsorbed to the surface of BLM via diffusion and gradually entered BLM through complicated changes in structure; the resistance of BLM is also reduced accompanying variations of the capacitance and the charge density at the surface of the membrane. So the current in the voltammograms becomes large (curve 2 in fig. 2 ) . The voltammetric characteristics of the dye are fully concurrent with those of Nile blue A and Toluidine blue 0 in ref. [5] and there is no peak appearing at the potential windows It- 150 mV. As this type of BLM can withstand less than 200 mV without breaking, peak might occur beyond the potential windows. This transport shows an irreversible property. Figure 3 is the cyclic voltammetric behavior of MB transport across different BLMs formed from three different types of lipids. The current of BLM formed from NPC is the largest and the current of BLM formed from PG is the smallest. These results can be explained by the variation of the structure and the charge density at the surface of the membrane caused by different types of membrane-forming lipids. In the BLM formed from NPC + OCH, BLM suffers the condensed effect and the membrane also is in good order due to the addition of OCH'~'. The permeability of
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E/(mV,
vs.
E/(mV, vs. SCE)
WE)
Fig.2 .
Characteristic cyclic voltammograms of MB transport acrasa BLM. pH= 5 . 2 2 . scan rate v = 10 rnV/s. 1, With-
out
MB; 2,
10 6 m o l / ~MB in cis-sidc..
Fig. 3 . Cyclic voltammograms of MB transport across BLM fornred from different lipids. 1 0 ~ 6 m o l / Lin cis-side, pH = 4.97, scan rate v = 5 0 mV/s. 1, NPC; 2 , NPC+ OCH;3,
PG.
the membrane to positively charged ions is sharply reduced by the above e f f e c d 9 ] . MB is a positively charged ion in an acidic solution, .so the transport of MB across the BLM is inhibited. PG is a negatively charged lipid, and it will combine with proton in the acidic solution and will bear positive charges at the surface of membrane. The transferring of a positively charged molecule, MB, would be inhibited. At the same time, the conductivity of other inorganic ions is poor in the membrane (unmodified BLMs are not good conductors to inorganic ions"'). So the current of this membrane is the smallest. In the B14M formed from NPC + OCH, according to these results obtained from X-ray and other experiments by Franks and l,ieb['O1, the P-OH group on the 17th carbon of cholesterol is located very close to the position of fatty acyl ester groups of lipid molecules. Although there is uncertainty of 1 . 5 carbons due to the difference in the vertical location of a- and Pchains, cholesterol with the rigid ring structure of penetrating into , diagram of oxidked the membrane of the 7-10th carbons of the alkyl chain and alkyl- ~ i 4 . ~ schematic chain tail of cholesterol reaches the 12-15th carbons. The only cholesterol in BLM. A, Lipid difference of oxidized cholesterol from cholesterol is the substitut- molecule* B* oxidized ed carbonyl group or hydroxyl group and the interaction of OCH cholesterol. with the headgroup of lipid which is stronger than that of cholesterol owing to the fact that the dipole movement of OC,H is larger than that of cholesterol. Combining with refs. [ l l , 121, the structure of OCH in BLM can be expressed in fig. 4. NPC is a mixture of different types of lipids with different lengths in the alkyl chains, but the headgroup is the same and is a neutral lipid at its isoelectric point. MB is a type of dye with certain lipophilic property, so it can be easily transported through the BLM formed from NPC and the current is the largest. 2 . 2 Voltammetric characteristics at different dye concentrations At the situation of I3I.M formed from NPC, the concentration of MB in the aqueous solution
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is changed. Fig. 5 shows that the cathodic and anodic currents all increase with the increase of the MB concentration, but no peak appears. This indicates that MB molecules can transfer across BLM and also can transmembrane from trans-side to cis-side; the peak might appear beyond the potential window and the transport is an irreversible one.
2 . 3 Voltammetric characteristics of MB modified-BLM under different aqueous solution pH ~ is added to the As shown in fig. 6, in the BLM formed from NPC, when 1 0 - ~ m o l /MB cisside of membrane under different aqueous solution pH, the transferring properties of MB across BLM are analyzed.
E/(mC', vs. SCE) Fig. 5 . Cyclic voltammograms of MB transport through BLM formed from NPC at different h4B concentrations. p H = 4.97,
E/(mV, vs. SCE) Fig. 6 . Cyclic voltammograms of BLM at different pH.
BLM
MB
transport through
was formed from
NpC,
scan rate v = 50 mV/s. MH added to trans-side. 1. ~ O - ~ m o l / mol/L in cis-side, scan rate v = 10 mV/s. 1, pH= 4.97;
L; 2, 10-'mol/~.
2 , p H = 4 . 1 2 ; 3 , p H = 3 . 0 5 ; 4, p H = 2 . 0 6 (curves3 and
4 are overlapped together and curve 4 is not shown here).
From fig. 6 we know that the current is reduced with the reduction of the aqueous solution pH. When pH value is less than 3, the current is extremely small. This behavior can be explained by taking pH-dependent factors into consideration. These factors include[51: ionic adsorption, isoelectric point of phospholipid, ionic transport of H + or O H - ions. MI3 is positively charged ion in the acidic solution. NPC is a neutral lipid and BLM formed from the lipid is a neutral one at its isoelectric point. The decrease of pH may result in reduced adsorption OH ions to the BLM surface. A positive charge will appear in its hydrophilic region when pH is reduced to overpass the isoelectric point of the lipid (ca. p H = 5. 5[13'). The transport of the positively charged MB across B1.M would experience a large electric repulsion at this time. So the transport of MB is inhibited when the aqueous soltuion pH is reduced. When pH value is less than 3, the resistance of BI,M is larger than 1 0 l O nand the ionic current only has about 10 PA. The current is equivalent to several decade ions transport across BLM per second and the membrane can be regarded as an insulator at this time.
3 Conclusion T h e voltammetric characteristics of MB transport across BI,M are concurrent with the first type of dyes in ref. [ 5 ] . There is no peak appearing in the range of potential window and
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the transferring can be regarded as an irreversible one. The transport is tightly related to the structure of the membrane. Oxidized cholesterol and lipid negatively charged inhibit the transport of MB. The pH of the aqueous solution also influences the transport. When the pH of the aqueous solution is close to the isoelectric point of the lipid, the voltammetric response is obvious. When the pH is lower than that of the isoelectric point, the transport will be inhibited. These results can be explained by considering the appearance of net positive charges at the surface of the membrane at lower pH. The above investigations can provide important information for further studying the catalysis of MB to some certain bio-active substances (such as NADH) at the surface of biomembranes (Received March 11, 1996)
References Tien, H. T . , Bilayer Lipid Membranes ( B L M ) : Theory and Practice, New York: Marcel Dekker, 1974. Fendler, J. H . , Membrane Mimetic Chemistry, New York: Wiley, 1982. Fendler, J. H . , Atomic and molecular clusters in membrane mimetic chemistry, Chem . R e v . , 1987, 87: 877. Ottava-Leitmannova, A. , Tien, H. T . , Bilayer lipid membranes: an experimental system for biomolecular electronic &vices development, Progress in Surface Science, 1992, 41 : 337. Kutnik, J . , Tien, H. T. , Cyclic voltammetry of dye-modified BLMs, Bioelectrochem . Bioenerg. , 1986, 16: 435. Kutnik, J . , Tien, H. T . , Application of voltammetric techniques to membrane studies, J . ' ~ i o c h e m .Biophys. Methods, 1985, 11: 317. Tien, H. T. , Mueller, D. , Rudin, D. 0. et al . , Reconstitution of cell membrane structure in vitro and its transformation into an excitable system, Nature, 1962, 194: 979. Kumar, V. V . , Anderson, W. H . , Thompson, E. W. et a l . , Asymmetry of lysophosphatidylcholine/cholesterol vesicles is sensitive to cholestrol modulation, Biochem . , 1988, 27: 393. Krull, U. J . , Planar artificial biomembranes optimized for biochemical assay, Anal. Chim . Acta, 1987, 197: 203. Franks, N. P . , Lieb, W. R . , The structure of lipid bilayers and the effects of general anesthetics, An X-ray and neutron diffraction study, J . Mol. Biol., 1979, 133(4) : 469. Pasenkiewicz-Gierule, M . , Suczyski, W. K . , Kusumi, A. , Rotational diffusion of a steroid molecule in phosphatidylcholinecholesterol membranes: fluid-phase microimmiscibility in unsaturated phosphatidylcholine-cholesterol membranes, Biochem . , 1990, 29: 4059. Subczyski, W. K.. Antholine, W. E., Hyde. J. S. et a1 . , Microimmiscibility and three-dimensional dynamic structure of phosphatidylcholine-cholesterol membranes: translational diffusion of a copper complex in the membrane, Biochem . , 1990, 29: 7936. Phillips, M. C . , Chapman, D. , Monolayer characteristics of saturated 1.2-diacyl phosphatidylcholines (lecithins) and phosphatidylethanolamines at the air-water interface, Biochim. Biophys. Acta, 1968, 163(3): 301. Acknowledgement The authors appreciate the donation of the (3-1087 computer-controlled electroanalytical systems from Prof. T . Kuwana (University of Kansas). This work was supported by the National Natural Science Foundation of China (Grant NO. 29392603).
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February 1997