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Bioluminescence of Monolayers of Firefly Luciferase Immobilized on Graphite S. Palomba,* N. Berovic, and R. E. Palmer Nanoscale Physics Research Laboratory, School of Physics and Astronomy, The UniVersity of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. ReceiVed March 4, 2006. In Final Form: March 25, 2006 We report on the immobilization of the firefly protein luciferase on the hydrophobic surface of graphite. Observation by liquid-phase atomic force microscopy of islands with a height consistent with the size of a single molecule confirmed that the protein was contained within a monomolecular layer. The enzyme activity was assayed by single-photon counting of the bioluminescence, which is the catalytic product of luciferase. Attachment to the surface modified the efficiency of the enzyme, but the introduction of the substrates luciferin and ATP resulted in the reactivation of the enzyme. The functionalized graphite surface was employed as a cathode in a bioelectrochemical cell. This demonstrated that the electric field caused a substantial loss of enzyme catalytic activity.
Introduction The immobilization of single protein molecules on solid surfaces is receiving great attention, motivated by interest in fundamental studies of protein function and conformation1,2 as well as possible applications in biosensors.3-7 Single-molecule measurements on surfaces typically require the immobilization of the protein molecule onto a surface. In recent work, we demonstrated that, for some proteins, the attachment to a surface demands the presence of gold or nickel clusters pinned to a graphite surface. 8-12 We found that the firefly enzyme luciferase forms monolayers of dispersed molecules on freshly cleaved graphite surfaces. This enabled us to address two fundamental questions of relevance to the construction of biosensors. (A) Does the immobilization of isolated luciferase molecules on hydrophobic graphite surfaces result in a loss of enzymatic activity? (B) Can the enzymatic activity be modulated with external signals (e.g., an electric field)? This latter example has a bearing on the study of the mechanisms behind protein functions. Luciferase, in common with all enzymes, operates in a narrow range of pH values. Earlier work13 has shown that when luciferase was immobilized within a layer of a few micrometers on the cathode of an electrochemical cell, the application of cathode volts caused a nonequilibrium shift of pH toward more alkaline values of pH, which was reflected in the change of the rate of (1) Branden, C.; Tooze, J. Introduction to Protein Structure; Taylor and Francis: London, 1999. (2) Hunte, C.; Michel, H. Curr. Opin. Struct. Biol. 2002, 12, 503. (3) Brusova, Z.; Ferapontova, E. E.; Sakharov, I. Y.; Magner, E.; Gorton, L. Electroanalysis 2005, 17, 460. (4) Wang, H. S.; Pan, Q. X.; Wang G. X. Sensors 2005, 5, 266. (5) Murata, M.; Yano, K.; Kuroki, S.; Suzutani, T.; Katayama Y. Anal. Sci. 2003, 19, 1569. (6) Fan, C.; Pang, J.; Shen, P.; Li, G.; Zhu D. Anal. Sci. 2002, 18, 129. (7) Zhang, W.; Li, G. Anal. Sci. 2004, 20, 603. (8) Leung, C.; Xirouchaki, C.; Berovic, N.; Palmer, R. E. AdV. Mater. 2004, 16, 223. (9) Prisco, U.; Leung, C.; Xirouchaki, C.; Jones, C. H.; Heath, J. K.; Palmer, R. E. J. R. Soc. Interface 2005, 2, 169. (10) Collins, J. A.; Xirouchaki, C.; Palmer, R. E.; Heath, J. K.; Jones, C. H. Appl. Surf. Sci. 2004, 226, 197. (11) Di Vece, M.; Palomba, S.; Palmer, R. E. Phys. ReV. B 2005, 72, 073407. (12) Palmer, R. E.; Pratontep, S.; Boyen, H.-G. Nat. Mater. 2003, 2, 443. (13) Chittock, R. S.; Glidle, A.; Wharton, C. W.; Berovic, N.; Cooper, J. M.; Beynon, T. D. Anal. Chem. 1998, 70, 0, 4170.
enzyme catalytic activity. The change was fully reversible with a time constant on the order of 100 s. This observation suggested that the local electric field due to ionized residues has a direct effect on the native active enzyme conformation. In the case of the enzyme subtilisin BPN, it appears that a genetically modified protein that had one of its ionizable residues removed had a distinctly lower efficiency even though the residue was in the region of the molecule far from the active site.14 Thus, the Coulomb field at intermediate range can influence the enzyme catalytic activity. In this article, we show that exposing the graphite surface to a dilute solution of firefly luciferase leads to the formation of a strongly bound protein monolayer on the surface. Wiping the protein layer with the AFM tip does not release the protein from the surface but instead forces the protein molecules to aggregate. The activity of the enzyme is assayed by detection of the bioluminescence, which is the product of the catalytic action of luciferase. Data reported here shows that the enzyme is affected both by the attachment to graphite and by the electric field. Experimental Section AFM imaging was done on a Nanoscope III (Digital Instruments) using etched Si tips. The scanning probe image processor (SPIP, Image Metrology) program was used for image processing. The graphite samples were monochromatic-grade highly ordered pyrolytic graphite (GE Advanced Ceramics). Samples were cut to form 10 mm × 5 mm × 0.6 mm sections, which were cleaved to expose a fresh surface. To this surface, 50 µL of a 1 µM solution of luciferase was applied. The solution contained luciferase (Luciola Mingrelica, Sigma-Aldrich L4899) in 25 mM HEPES buffer, adjusted to pH 7.8. The protein was allowed to settle for 20 min at 2 °C, after which the surface was rinsed with pure HEPES buffer solution. To assay bioluminescence, the sample was transferred into a PTFE container and immersed in the solution containing 500 µM D-salt luciferin (Sigma-Aldrich L6882), 8 mM ATP and Mg2Cl, and 25 mM HEPES adjusted to pH 7.8. Luciferin is converted by the enzyme into a dioxetanone form, which decays by the emission of a photon in a band centered on 550 nm. An acrylic optical fiber (2 mm ESKA) was immersed in the solution facing the protein-coated surface. The output from the fiber was optically coupled with silicone fluid to the photocathode of a channel photomultiplier module (CPM 982, Perkin-Elmer). Detected photon pulses were counted in a multiscaler (14) Jackson, S. E.; Fersht, A. R. Biochemistry 1993, 32, 13909.
10.1021/la060597h CCC: $33.50 © 2006 American Chemical Society Published on Web 05/11/2006
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Figure 2. (a) Liquid-phase deflection mode AFM image of a firefly luciferase monolayer on graphite, collected after sweeping the central area (1 µm × 1 µm) with the AFM tip. (b) Diameter distribution confirms a monodispersed layer of luciferase on the surface with an average value of 37.7 ( 13.2 nm. Taking into account the convolution with the tip, the dimensions are reasonably consistent with the X-ray protein structure. A graphite sample was connected as a cathode of a three-electrode electrochemical cell. A copper wire was attached to the graphite with silver paste and covered by an insulating layer of nail varnish. The reference electrode was Ag/AgCl, and the counter electrode was a Pt-Ir wire. An I-V measuring system (Keithley 236) was used to apply a current and measure the voltage of the cathode relative to the reference electrode, controlled by a LabView 7 program written for the purpose.
Results and Discussion Figure 1. (a) Liquid-phase AFM image of a firefly luciferase monolayer adsorbed on graphite. This topographic image shows the compact single monolayer formed by the protein. (b) Height distribution for the AFM image depicted in Figure 1a, together with a Gaussian fit. The mean height of about 10.4 ( 3.62 nm is reasonably close to the size of a single protein. (c) Diameter distribution of the AFM image depicted in Figure 1a, together with a Gaussian fit. It shows an average size of about 41.7 ( 16.3 nm. module (TurboMCS, ORTEC). The optical system was calibrated using a beta light containing tritium (H-3) with a nominal activity of ∼1.4 GBq and using a phosphorescent rubber that has a slow exponential decay comparable to luciferin-luciferase bioluminescence.
Figure 1a shows a topographic liquid-phase AFM image of luciferase on graphite. The organization of the protein molecules into a single layer adsorbed on the carbon surface is evident. A detailed analysis of the image using SPIP confirms this conclusion. Figure 1b and c shows the height and lateral distributions corresponding to Figure 1a; the Gaussian fit reports a mean height of 10.4 ( 3.62 nm and an average diameter of 41.7 ( 16.3 nm. This compares well with the luciferase unit cell in the crystalline state, obtained by X-ray diffraction, which is 11.9 nm × 11.9 nm × 9.5 nm.15 Figure 2a shows the effect of “wiping” an area of 1 µm × 1 µm with the AFM tip applying a force of about 7 nN. This (15) Conti, E.; Franks, N. P.; Brick, P. Structure 1996, 4, 287.
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Figure 3. Bioluminescence intensity collected from monodispersed luciferase on graphite over about 1 h. The exponential fit confirms the natural decay of the enzymatic activity.
Figure 5. (a) Bioluminescence quenching, as in Figure 4, due to the stepwise increase in application (b). Note the slight recovery of the bioactivity between the first and the second current steps (circled) due to the presence of about four protein monolayers.
Figure 4. (a) Bioluminescence quenching after applying the current pulse (-100 µA) depicted in b. The arrows in the main picture indicate the start and end of the pulse. Note that the bioactivity was not recovered after the end of the applied pulse.
exposed the underlying graphite surface while proteins were pushed into forming larger clusters. Figure 2b displays the lateral distribution corresponding to Figure 2a. The average diameter is 37.7 ( 13.2 nm. To assay the bioactivity, the sample was transferred into a PTFE container, and then the solution with substrates described above was applied. Luciferase reacts according to MichaelisMenten kinetics; therefore, the concentrations used were well in
excess to reach the saturation region.16 Figure 3 shows the light output recorded immediately after 150 µL of substrate solution was applied to the immobilized protein. The initial period of 1000 s displays an increase in the count rate from 30 to 120 cps. By contrast, we observed that when assayed in bulk solution the enzyme exhibits a rapid rise followed by a steady exponential decline due to the accumulation of the products of reaction that inhibit the activity of the enzyme, as reported in the literature.17 Comparing the catalytic rates of luciferase in solution and when it is attached to graphite, we observed that the slow rise of about 1000 s, as depicted in Figure 3, is present only when the protein is bound to the graphite surface. Therefore, attachment to the hydrophobic surface has evidently inhibited the enzyme. We assume that the reactivation over the period of 1000 s has been caused by the presence of substrates. Figure 4a shows the effect of a 0.1 mA current pulse applied to the cathode when luminescence was in close proximity of the maximum. Figure 4b shows the corresponding cathode volts. The consequence to the bioluminescence was a decline by 2/3 of its intensity. No recovery, after the pulse had been removed, is in evidence. When a similar protocol was performed on a sample (16) Lembert, N.; Idahl, L. A. Biochem. J. 1995, 305, 929. (17) Lemasters, J. J.; Hackenbrock, C. R. Biochemistry 1997, 16, 445.
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that had four layers of protein molecules (obtained by deposition from a more concentrated stock solution), a similar result was obtained as shown in Figure 5a. Figure 5b shows that current was removed at 800 s and subsequently opposite-polarity pulses were applied, making the graphite an anode. No significant recovery was seen during this procedure. It is possible that small degree of recovery occurred when the cathode current was switched off (indicated by the circle in Figure 5a). The responses to an electric field obtained here are very different from those observed when the protein was contained within a layer several micrometers from the cathode.13 In the present case, the close proximity to the graphite surface placed the protein molecules within the electrical double layer, which is on the order of 300 Å.18 The electric field strength in this position could be as high as 100 MV/m, which is strong enough to affect the bioactivity of the enzyme. The consequence for the enzyme is irreversible damage caused either by the field or by cathodic deprotonation. For the sample with four protein monolayers, an increase in thickness does not alter the fact. The main effect of having protein stacked in four layers is that the response times are faster during both reactivation and cathodic denaturing. Because the time of transport through four layers is longer than through a monolayer, (18) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; Wiley: New York, 2001.
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it does not seem that electron or ion transport is the ultimate cause of the decrease in bioluminescence. The close proximity of larger numbers of protein molecules in this sample is the significant difference from the sample with a single monolayer. This is responsible for a faster response of bioluminescence as a comparison between Figures 4a and 5a shows.
Conclusions To answer the questions posed at the outset, we have produced a monolayer of luciferase on a hydrophobic graphite surface. Both AFM images and the effects of wiping confirm that. However, attachment resulted in a reversible degradation of the catalytic efficiency of the enzyme. This impairment could be reversed by the application of substrates ATP and luciferin. When an electrical current was applied, which results in the discharging of H+ ions from the solution on the graphite surface, 75% of the luciferase catalytic activity was lost. Both processes, reactivation with substrates and cathodic denaturing, produce a slow response of bioluminescence on the scale of minutes. These response times become significantly shorter when the graphite surface is functionalized with four layers of protein instead of a monolayer. Acknowledgment. This work was supported by EPRSC and the EU Research and Training Network “Nano Cluster”. LA060597H
