J. Phys. Chem. C 2009, 113, 14377–14380
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Insertion of a Cytochrome c Protein into a Complex Lipid Monolayer under an Electric Field Hwajeong Kim,†,‡ Patrick Degenaar,‡ and Youngkyoo Kim*,† Organic Nanoelectronics Laboratory, Department of Chemical Engineering, Kyungpook National UniVersity, Daegu 702-701, Republic of Korea, and Institute of Biomedical Engineering (IBE), Imperial College London, London SW7 2AZ, United Kingdom ReceiVed: March 18, 2009; ReVised Manuscript ReceiVed: June 18, 2009
Here we report the insertion of cytochrome c into lipid monolayers made with a mixture of phosphatidic acid (PA), which is an essential platform for mimicking part of the electron transport chains in mitochondria. The monolayer of the PA mixture was prepared on a gold substrate by the Langmuir-Blodgett technique. The insertion and migration behavior of cytochrome c with the artificially made lipid monolayer of the PA mixture has been investigated by measuring the current-potential characteristics. Results showed that cytochrome c was successfully inserted into the monolayer of the PA mixture as evidenced from the surface images exhibiting obvious domains of cytochrome c under varied potential (electric field). The maximum amount of cytochrome c inserted into the monolayer of the PA mixture was calculated to be 155 ng/0.2 cm2. Introduction Recently, artificial organs, which can replace eye, cochlear, heart, liver, lungs, pancreas, and kidney implants, have attracted keen interest because of the possibility of extending the average span of human life.1-3 For the successful operation of artificial organs implanted in a human body, however, their biocompatibility should be secured and a sophisticated energy source must be invented for continuous working of the organs. In previous works, a series of devices such as biofuel cells, micromotors, and solar cells have been attempted as the energy source for artificial organs.1,4-7 Unfortunately, these devices were insufficient for practical applications because of their low efficiency and unsatisfied biocompatibility of materials used. Hence, to date, it is strongly considered that the best realistic way is the exploitation of energy in real living systems such as electron transport chains in mitochondria. Bioenergetics (transformation of energy in living systems) is known to be based on the production and destruction of adenosine triphosphate (ATP) molecules (a platform for generating chemical energy), together with a variety of oxidationreduction reactions.8,9 ATP is made from adenosine diphosphate (ADP) through a series of electron transfer and H+ diffusion processes in the respiratory complex inside mitochondria, which are the so-called cellular power plants in aerobic respiration.8,9 The respiratory complex consists of complex I, II, III, IV, and cytochrome c (cyt c) as an electron transfer mediator. Here cyt c, which normally resides over both inner-membrane and intermembrane spaces in mitochondria and is bound to acidic phospholipids and cardiolipin (CL) via electrostatic and/or hydrophobic interactions, functions as an electron shuttle between complex III and complex IV via cyt c oxidase and reductase.10 Of various essential proteins in the electron transport chain inside mitochondria particularly, cyt c has been extensively * Corresponding author. E-mail:
[email protected] or y.kim@ imperial.ac.uk. † Kyungpook National University. ‡ Imperial College London.
studied in the fields of bioelectronics (biofuel cells, biosensors, etc.) because of its efficient electron transfer characteristics by iron heme.11-14 Most of these studies [electron transfer rate, reversibility, and potential window (reduction-oxidation) investigations] have been carried out with cyt c that is immobilized on chemically modified materials such as conducting polymers, self-assembled monolayers with thiol groups, inorganic compounds (titania), and lipids.11-17 Electron transfer and interaction studies have also been reported on cyt c that is bound to CL or phosphatidylcholine or phosphatidic acid (PA)-modified layers.16,17 However, PA (one of acidic phospholipids) in mitochondria has a complex structure that consists of alkyl chains with single and/or double bonds as well as various chain lengths. Hence, considering the real living system where cyt c exists, these previous studies have a fundamental limitation because of a lack of mimicking the exact environment. In this work, in order to mimic one of the real lipids in living systems, we attempted to make an acidic phospholipids monolayer using a mixture of various PAs that contain alkyl chains with single (saturated) and double (unsaturated) bonds as well as various chain lengths. The complex PA mixture used here consists of dipalmitoyl (C16:0) PA (DPPA), distearoyl (C18: 0) PA (DSPA), dioleoyl (C18:1) PA (DOPA), and dilinoleoyl (C18:2) PA (DLPA) (Figure 1, left). The monolayer of the PA mixture was prepared by the Langmuir-Blodgett (LB) technique (Experimental Section). Here, Au substrates were employed as an electrode to monitor electron transport characteristics either from or to the cyt c molecules through the monolayer of the PA mixture. Then the insertion and migration behavior of cyt c with these artificially made lipid monolayers has been investigated by measuring the current flow and extent of insertion in order to mimic part of the electron transfer chain reactions in mitochondria (Figure 1, right). Experimental Section Materials. A phosphate buffer solution (PBS, 0.1 M, pH 7.0) was prepared from monosodium phosphate and disodium phosphate, while high purity deionized water (18 MΩ) was prepared using a water distillation system (Millipore). Horse
10.1021/jp902450g CCC: $40.75 2009 American Chemical Society Published on Web 07/16/2009
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Kim et al.
Figure 1. Schematic illustration of the insertion of cyt c into the monolayer of complex lipids (phosphatidic acid mixture) that is coated on a Au substrate (see Experimental Section for names of lipid).
heart cyt c that was purchased from Sigma Chemical Co. was converted to a fully oxidized form by the reaction with excess K3Fe(CN)6, followed by purification with ion exchange chromatography using a Whatman CM-32 column and then elucidated with 0.50 M NaCl and a 10 mM phosphate buffer solution (pH 7.0).18 Eluent containing the purified protein was concentrated by ultrafiltration using Amicon YM-3 membranes, dialyzed extensively to remove phosphate, and used to make aqueous solutions using PBS.18 A mixture of various phosphatidic acids (PAs) was purchased from Sigma Chemical Co. Here, we note that the (PA) mixture contains dipalmitoyl (C16:0) PA (33%), distearoyl (C18:0) PA (13%), dioleoyl (C18:1) PA (31%), dilinoleoyl (18:2) PA (15%), and sodium ions (8%). For the LB experiments, the PA mixture solutions were prepared using chloroform at a concentration of 1 mg/mL. Measurements. We employed Au substrates (9 MHz ATcut) which feature a Au thickness of ∼300 nm and an area of 0.2 cm2. The PA mixture solution was poured on top of a subphase (DI water) in the trough of the LB system (NIMA 611, U.K.). Then, the solvent (chloroform) molecules were removed by natural evaporation for 20 min at room temperature. Next, the PA mixture was compressed at a force of 38 mN/m, leading to the monolayer of the PA mixture, and was then transferred onto a Au substrate at a deposition rate of 2 mm/ min at room temperature. The deposition ratio of monolayer was 1.02 ( 0.05. Atomic force microscopy (AFM) (multimode system, Nanoscope IIIa, Veeco Co. Ltd., U.S.A.) was employed to measure the surface of monolayers. Basic electrochemical experiments were carried out using a potentiostat/galvanostat system (EG&G Princeton Applied Research model 273A), while electrochemical quartz crystal microbalance (EQCM) experiments were performed using a potentiostat/galvanostat system equipped with a quartz crystal analyzer (SEIKO EG&G model QCA 922). Reference and counter electrodes used were Ag/ AgCl (in saturated KCl) and Pt wire, respectively. Results and Discussion The surface pressure-area (π-A) isotherm of the present PA mixture showed a typical shape that is frequently observed for lipids (Figure 2a).19 Upon moving the barrier in the LB system, the lipid molecules of the PA mixture at the air-water interface were compressed and reached a critical point (liquid-solid transformation) at a surface pressure of ∼35 mN/m. The formation of a solid lipid monolayer was confirmed at 37 mN/ m, in which the mean area per molecule was 95 Å2. DPPA and
Figure 2. (a) Surface pressure-area (π-A) isotherm of PA mixture at the air-water interface. (b) AFM images of the monolayer of the PA mixture coated on a Au substrate at a pressure of 41 mN/m (top) and 38 mN/m (bottom).
DSPA were reported to exhibit a steeper π-A curve slope (liquid phase) leading to a more condensed phase because they have only single-bonded alkyl chains (tail part),13,14 even though their mean area does also depend on the size of diacyl and phosphate groups in their head part. In the present PA mixture, however, the slope of the π-A curve seems to be slightly more gentle, which can be attributed to the kinked structure of DOPA and DLPA because of the double-bonded alkyl chains.20 From the π-A curve, we confirmed that the solid PA monolayer was formed at 38-43 mN/m, while the monolayer collapsed at >43 mN/m. Figure 2b shows the atomic force microscopy (AFM) images of the PA monolayers, which were transferred to Au substrates at 41 mN/m (top) and 38 mN/m (bottom), respectively. In the case of 41 mN/m, the average particle size was
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Figure 4. (a) EQCM curve [frequency change versus potential at a (potential) scan rate of 10 mV/s] and (b) QCM curve (frequency change versus time at an applied potential of 0.1 V) for the insertion of cyt c into the monolayer of the PA mixture coated on a Au substrate in the 0.1 M PBS solution that contains 0.1 mM cyt c. Inset is illustration of the EQCM measurement system. Figure 3. (a) Current-potential (redox) curve for the insertion of cyt c into the monolayer of the PA mixture in the 0.1 M PBS solution that contains 0.1 mM cyt c at a (potential) scan rate of 10 mV/s. (b) AFM images of cyt c inserted into the monolayer of the PA mixture at an applied potential of 0.6 V (top), 0.15 V (middle), and -0.1 V (bottom).
∼18 nm in the presence of bigger particles (>37 nm), which can be attributed to the partially collapsed molecules because of the higher pressure. In contrast, the surface of the monolayer transferred at 38 mN/m was relatively more uniform leading to the average particle size of 8 nm. This trend is clearly observed in the three-dimensional (3D) images. Next, we performed the insertion of cyt c into the monolayer of the PA mixture coated on Au substrates in the phosphate buffer solution (PBS, 0.1 M) by applying a potential to the Au substrates (Experimental Section). Considering the positive net (surface) charge of cyt c, a negative bias is required to insert cyt c molecules into the monolayer. As shown in Figure 3a, this negative bias direction corresponds to the reduction process (we note that the present cyt c supplied is an oxidized form initially, so that the first scan should be a reduction process to monitor the oxidation peak). The reduction (Fe3+ f Fe2+) peak was observed at 0.03 V versus Ag/AgCl in a saturated KCl solution, while the oxidation (Fe2+ f Fe3+) peak of cyt c was measured at 0.31 V. This result informs us that the redox process proceeded quasi-reversibly with the potential difference of ∆EP ) 0.28 V, indicating that cyt c did not directly exist on the surface of the Au electrode, but it was away from the Au surface at a distance (we note that ∆EP should be less than 0.059 V, if cyt c did directly contact the Au surface).14,22 Hence, cyt c is considered to be “inserted” into the lipid layer and not contacting the Au surface, which is very similar to its existence in the
electron transfer chain in mitochondria.8-10 This is obviously observed from the AFM images (Figure 3b). To further understand the location where cyt c is inserted in the monolayer of the PA mixture, we measured a couple of AFM images by varying the potential in the reduction direction. As shown in the top panel of Figure 3b, at the applied potential of 0.6 V, no cyt c was measured in the lipid layer, which can be attributed to the insufficient positive potential for cyt c (see far away from the oxidation peak in Figure 3a). Here, we need to pay particular attention to this surface image because it proves that there is a strongly bound lipid layer on the Au substrates, even though high potential was applied. This reconfirms the justification of this study in the environment of an aqueous medium and under applied potential. When the applied potential is 0.15 V, a few cyt c domains were observed, indicating initialization of the cyt c insertion process (Figure 3b, middle). Here, we note that this potential is the onset voltage of cyt c reduction (Figure 3a). At this stage, the size of cyt c domains was 85-120 nm. Upon further decrease in the potential to -0.1 V, the population of cyt c domains was increased as shown in the bottom panel of Figure 3b. In addition, the domain size was also increased to ∼160 nm. It is noteworthy that this actual image resembles the schematic illustration in Figure 1.8,9 When it comes to the smallest size (∼85 nm) of cyt c measured in the AFM images, it is ∼25 times larger than the size (3.4 nm) of single cyt c.23 This discrepancy can be attributed to the aggregation of cyt c due to the high concentration of cyt c (0.1 mM) when inserted into the monolayer of the PA mixture. We note that in our previous study it was possible to make ∼12 nm sized cyt c domains on a conducting polymer layer because of the covalent bond formation between the lysine groups of cyt c and functional groups of the conducting polymer.24 However, we think that making smaller cyt c domains is not easy because of the repulsive interaction between the hydro-
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phobic tail groups of lipids and hydrophilic protein surfaces of cyt c. It is noteworthy that the insertion process was enabled because of the attractive interaction between the positive charge (NH3+) of the lysine group in cyt c and negative charge of the headgroup (PO4-) in lipids at the initial stage of insertion. To study the amount of cyt c that is inserted into the monolayer of the PA mixture, we employed the electrochemical quartz crystal microbalance (EQCM) technique in combination with the QCM technique.22,25 The scanned potential range was limited to the reduction part (negative potential to the Au substrate) that is related to the actual insertion of cyt c. As shown in Figure 4a, the frequency change (∆F) was initially oscillated and then greatly decreased, indicating the increased insertion of cyt c as observed in the AFM images (Figure 3b). The increased insertion of cyt c at the fixed negative bias (reduction) was also monitored from the QCM data with time (Figure 4b), which confirms that cyt c was indeed inserted into the monolayer of the PA mixture. From the ∆F data, we calculated the maximum amount (∆m) of the inserted cyt c molecules into the monolayer of the PA mixture using the modified equation from the Sauerbrey relation:25,26 ∆F ) -2F02∆m/A(Fqµq)1/2 ) -K∆m, where ∆F is the frequency change caused by addition of mass per unit area, ∆F0 is the fundamental crystal frequency, A is the electrode surface area, Fq is the density of quartz, and µq is the shear modulus of quartz. The constant K in the equation was determined using AgNO3 as indicated in the manual of the EQCM system (Experimental Section). The calculated maximum amount of the inserted cyt c into the monolayer of PA mixture was ∆m ) 155 ng/0.2 cm2 at ∆F ) 14 Hz (-0.1 V). Conclusions In summary, a lipid monolayer of a PA mixture coated on Au substrates was prepared using the LB technique. A series of AFM measurements in liquid and/or dry conditions proved that the monolayer of the PA mixture was strongly bound to Au substrates. The insertion of cyt c into the monolayer of the PA mixture was carried out in the PBS solution of cyt c (high concentration). The successful insertion was evidenced from surface images that showed obvious domains of cyt c by varying the applied potential. The maximum amount of cyt c inserted into the monolayer of the PA mixture was calculated to be 155 ng/0.2 cm2 from a EQCM measurement, while a QCM measurement confirmed the continuous insertion with time at a fixed reduction (negative) potential. The present successful insertion of cyt c into the monolayer of the PA mixture, which is one of the closet lipid structures to a real lipid system in mitochondria, may shed light on further research for mimicking
Kim et al. energy systems in living cells (such as a one-electron transfer between complex III and IV), leading to the invention of energy sources for artificial organs. Acknowledgment. This work was mainly supported by the Korean Research Foundation (KRF, Grant KRF-2006-214E00016) and partially by two Korean government projects (KOSEF-R01-2007-000-10836-0 and KRF-2007-331-D00121). References and Notes (1) Chow, A. Y.; Chow, V. Y.; Packo, K. H.; Pollack, J. S.; Peyman, G. A.; Schuchard, R. Arch. Ophthamal. 2004, 122, 460–469. (2) Clark, G. M. Phil. Trans. R. Soc. B 2006, 361, 791–810. (3) Galleti, P. M.; Nerem, R. M. Tissue Engineering and Artificial Organs, 3rd ed.; CRC Press; Inc.: Taylor & Francis, 2006; Section VI. (4) Mavroidis, C.; Dubey, A. Nat. Mater. 2003, 2, 573–574. (5) Cura, V. O. D.; Cunha, F. L.; Aguiar, M. L.; Cliquet, A. Artif. Organs 2003, 27, 507–516. (6) Kim, Y.; Cook, S.; Tuladhar, S. M.; Choulis, S. A.; Nelson, J.; Durrant, J. R.; Bradley, D. D. C.; Giles, M.; McCulloch, I.; Ha, C.-S.; Ree, M. Nat. Mater. 2006, 5, 197–203. (7) Davis, F.; Higson, S. P. J. Biosens. Bioelectron. 2007, 22, 1224– 1235. (8) Voet, D.; Voet, J. G.; Pratt, C. W. Fundamentals of Biochemistry: Life at the Molecular LeVel, 2nd ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2006. (9) Fox, S. I. Human Physiology 6th ed.; WCB/McGraw-Hill: Boston, MA, 1999. (10) Garrido, C.; Galluzzi, L.; Brunet, M.; Puig, P. E.; Didelot, C.; Kroemar, G. Cell Death Differ. 2006, 13, 1423–1433. (11) Willer, I. Science 2002, 298, 2407–2408. (12) Spricigo, R.; Dronov, R.; Ragagopalan, K. V.; Lisdat, F.; Leimkuhler, S.; Scheller, F. W.; Wollenberger, U. Soft Matter 2008, 4, 972–978. (13) Stoll, C.; Kudera, S.; Parak, W. J.; Lisdat, F. Small 2006, 2, 741– 743. (14) Fedurco, M. Coord. Chem. ReV. 2000, 209, 263–331. (15) Topoglidis, E.; Cass, A. E. G.; O’Regan, B.; Durrant, J. R. J. Electroanal. Chem. 2001, 517, 20–27. (16) Boussaad, S.; Dziri, L.; Arechabaleta, R.; Tao, M. J.; Leblanc, R. M. Langmuir 1998, 14, 6215–6219. (17) Hitotsumatsu, R.; Amao, Y. J. Photochem. Photobiol. B 2005, 79, 89–92. (18) Spooner, P. J. R.; Watts, A. Biochemistry 1991, 30, 3871–3879. (19) Bos, M. A.; Nylander, T. Langmuir 1996, 12, 2791–2797. (20) Ballet, P.; Auweraer, M. V.; Schryver, F. C. D.; Lemmetyinen, H.; Vuorimaa, E. J. Phys. Chem. 1996, 100, 13701–13751. (21) Chunbo, Y.; Ying, W.; Yueming, S.; Zuhong, L.; Juzheng, L. Surf. Sci. 1997, 392, L1–L6. (22) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2001. (23) Lvov, Y.; Arign, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117–6123. (24) Kim, H.; Lee, K.; Won, M.; Shim, Y. Langmuir 2008, 24, 1087– 1093. (25) Buttry, D. A.; Ward, M. D. Chem. ReV. 1992, 92, 1355–1379. (26) Sauerbrey, G. Z. Phys. 1959, 155, 206.
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