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2009, 113, 5027–5030 Published on Web 03/06/2009
Selective Degradation of Proteins by Laser Irradiation onto Gold Nanoparticles in Solution Yoshihiro Takeda,*,† Fumitaka Mafune´,‡ and Tamotsu Kondow§ East Tokyo Laboratory, Genesis Research Institute, Inc., 717-86 Futamata, Ichikawa, Chiba 272-0001, Japan, Department of Basic Science, Graduate School of Arts and Sciences, The UniVersity of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan, and Cluster Research Laboratory, Toyota Technological Institute, 717-86 Futamata, Ichikawa, Chiba 272-0001, Japan ReceiVed: October 24, 2008; ReVised Manuscript ReceiVed: February 11, 2009
Lysozyme and bovine serum albumin (BSA) were selectively degraded in their mixed solution containing gold nanoparticles by an appropriate pH selection of the solution under irradiation of a laser with 532 nm corresponding to the plasma oscillation of the nanoparticles. Lysozyme and BSA were selectively degraded at a pH of 7.0–11.0 and ∼4.0, respectively. The mechanism of the selective degradation is as follows: Lysozyme molecules are considered to aggregate selectively on the gold nanoparticle in a pH range of 7.0–11.0, because they have a sufficiently small net charge of an opposite sign to the ζ-potential of the gold nanoparticle. In this pH range, the lysozyme molecules aggregating on the gold nanoparticle are degraded selectively under the laser irradiation, because the gold nanoparticle creates a high temperature region (chemical species in this region is suffered from degradation) in its close vicinity by the laser irradiation as shown in our previous studies. The same discussion is true to the selective degradation of BSA at a pH of ∼4.0. When gold nanoparticles in an aqueous solution are irradiated by an intense laser with the wavelengths corresponding to the plasmon and the interband transitions, the photons are absorbed efficiently by the gold nanoparticles and converted into heat.1-3 The heat is transmitted to surrounding solvent molecules. As a result, a high temperature region is created in a close vicinity of the photoexcited gold nanoparticle.4,5 It is found that the high temperature region having a diameter as small as several hundreds nanometers is chemically reactive, while the reactivity diminishes in the outside of the region.6-8 In processing in biological and medical applications, this sharp molecular-scale confinement of the high temperature region has a greater benefit, which is not achieved by any other methods.9-13 In the present study, we succeeded selective degradation of proteins such as lysozyme and bovine serum albumin (BSA) in the high temperature region in the solution having an appropriate pH. The solution pH determines the net charges of the protein molecules and the ζ-potential of the gold nanoparticle and accordingly controls the protein-gold nanoparticle interaction and the protein-protein interaction. Let us consider the case that the net charge of the protein molecule has an opposite sign to the ζ-potential of the gold nanoparticle and in addition the net charge of the protein molecule is small. The protein molecule has a tendency of aggregation at the surface of this gold nanoparticle, and thus the protein molecule is selectively adsorbed on the gold nanoparticle. If a laser is irradiated there, the protein molecules in the high temperature region created by the laser irradiation are efficiently and selectively degraded. * To whom correspondence should be addressed. E-mail: takeda@ clusterlab.jp. † Genesis Research Institute, Inc. ‡ The University of Tokyo. § Toyota Technological Institute.
10.1021/jp809438d CCC: $40.75
An aqueous solution containing surfactant-free gold nanoparticles (0.6 nM, 21.5 ( 7.3 nm in diameter) was obtained as described previously.14-16 Lysozyme (the final concentration, 50 µg/ml, 3.6 µM) and BSA (the final concentration, 50 µg/ml, 0.75 µM) were added in the solution so as the weights of these proteins were same. The pH of the solution was changed from 12.0 to 2.0 by adding Tris buffer, ammonium acetate buffer, and KCl buffer (the final concentrations of these buffers, 100 mM). The procedures of the pulsed laser irradiation onto the solution and the determination of the amounts of the proteins in the solution were described previously.15,17 Panels a and b in Figure 1 depict SDS-polyacrylamide electrophoresis gels obtained from an aqueous solution of lysozyme and BSA after a pulsed laser irradiation. We also preformed an experiment for a solution containing BSA and lysozyme without gold nanoparticles. No proteins were found to be degraded. Panels c (pH 4.0) and d (pH 9.0) in Figure 1 show the degrees of degradation of the proteins, as a function of the time of the laser irradiation. The degrees of degradation of lysozyme and BSA (Deg(t)) are defined as
Deg(t) )
I(0) - I(t) I(0)
(1)
where I(t) represents the intensity of the protein band in the gel at an irradiation time, t min, and I(0) is the band intensity at t ) 0. In the repetitive degradation scheme,15,16 the time dependence of the degree of degradation should be expressed by the exponential function
f(t) ) 1 - exp(-t/τ) 2009 American Chemical Society
(2)
5028 J. Phys. Chem. C, Vol. 113, No. 13, 2009
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Figure 3. UV-vis absorption spectra of the mixed solution of BSA, lysozyme and gold nanoparticles measured before and after laser irradiation at the pH of 4.0 (a) and at the pH of 7.0 (b). Insets show the time of laser irradiation.
Figure 1. Panels a and b show pictures of an SDS-polyacrylamide electrophoresis gels of an aqueous solution of lysozyme and BSA dispersed with gold nanoparticles, which were irradiated with intense laser (532 nm, 17 mJ/pulse) for different irradiation times. The numbers on each lane of the pictures represent laser irradiation times. Arrows marked by L and B in the panels a and b indicate the positions of lysozyme and BSA, respectively, in the electrophoresis gels. Panels c and d show degradation degrees of lysozyme and BSA plotted against the laser irradiation times at the pHs of 4.0 and 9.0, respectively. The solid circles and the solid triangles indicate the degradation degree of lysozyme and BSA, respectively. The degradation degree of unity shows that lysozyme or BSA in the solution is fully degraded. The solid lines in panels c and d represent the fitting curves to the data by using eq 2.
Figure 2. Panels a and b represent degradation rates of lysozyme and BSA as a function of a solution pH, respectively. Filled circles and triangles represent the degradation rates of lysozyme and BSA in a solution with gold nanoparticles and without gold nanoparticles, respectively.
where 1/τ represents the degradation rate of the protein. Figure 2 shows the degradation rates, 1/τ of lysozyme (panel a) and BSA (panel b) as a function of the solution pH with the gold nanoparticles and without gold nanoparticles. The degradation rates, (1/τ) of lysozyme is high at pH of 7.0–11.0, while it is low at pH above 12.0 and below 6.0. On the other hand, the degradation rate of BSA is high at pH of 4.0 and is low otherwise. The highest degradation rate of BSA at pH of 4.0 is
about one-third of that of lysozyme at pH around 9.0. The errors in the degradation rates are due to the systematic errors of the measurement of the band intensity in the gel of the SDS electrophoresis and are the following: (1) The inhomogeneity of the size of the protein band in the gel due to the nonuniform distribution of the voltage applied on the gel for electrophoresis. (2) The stain of the gel after electrophoresis, which gives the errors in measuring of the band intensity. UV-vis absorption spectra of a mixed solution of BSA, lysozyme, and gold nanoparticles at the pHs of 4.0 and 7.0 were measured before and after laser irradiation as shown in Figure 3. The absorption decrement at ∼285 nm was observed when the BSA or lysozyme was degraded at pH 4.0 or 7.0, respectively. These results indicate that the indole and aromatic rings were degraded in both the cases of the lysozyme and the BSA degradation in the high temperature region. The absorption spectrum in the vicinity of 250 nm is contributed by an interband transition of a gold nanoparticle in the solution. The interband tends to be more intense as the gold nanoparticle size decreases.18,19 The absorbance increases with the time of laser irradiation as shown in Figure 3. This finding implies that the size of the gold nanoparticles decreases with the time of laser irradiation. The selective degradation of lysozyme and BSA is explained in terms of the ζ-potential of a gold nanoparticle and the net charge of a protein molecule concerned. At the first place, we describe the pH dependences of the ζ-potential and the net charge. The ζ-potential of surfactant-free gold nanoparticles should be negative in the entire pH range studied because several percents of gold atoms at the surface of the gold nanoparticle change to Au–OH.20,21 Luong and co-workers showed that the ζ-potential of gold nanoparticles should increase as the pH decreases below the pK of 5.8, and remained unchanged at minus several tens of millivolts above the pK.21 This pH dependence of the ζ-potential is consistent with the color change of a solution containing the gold nanoparticle observed in the present experiment. The color of the solution is wine-red at a pH above 7.5 and changed to dark purple below 6.9. The color change is caused by red shift of the absorption band of the gold nanoparticles due to their aggregation which has a close relation to the ζ-potential of the gold nanoparticles.22,23 The net charge of a protein molecule in a solution is positive at pH < pI (isoelectric point), neutral at pH ) pI, and negative at pH > pI;24-26 the pIs of lysozyme and BSA correspond to 11.0 and 4.7, respectively.27,28 Let us compare the pH dependence of the ζ-potential of gold nanoparticles and the net charge of proteins with that of the
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Figure 4. Schematic diagrams of a protein-gold nanoparticle and a protein-protein interactions in the solution with different pHs. Both directional arrows positioned between the proteins represent repulsion forces between the proteins.
selectivity of the degradation rates of the proteins as shown in Figure 4. In a pH above 12.0, both lysozyme and BSA are not degraded, because the net charges of lysozyme and BSA are negative and the ζ-potential of the gold nanoparticles is also negative and hence they do not interact with the gold nanoparticles. In a pH range of 7.0–11.0, the lysozyme molecule is positively charged and attached to the gold nanoparticle. In addition, the lysozyme molecules are slightly charged so that they tend to aggregate on the surface of the gold nanoparticle, while the BSA molecules are dissolved uniformly because the net charge of the BSA molecules is highly negative. Under these circumstances, the lysozyme molecules attached to the gold nanoparticle are degraded but the BSA molecules are not degraded (selective degradation). In a pH range of 5.0–6.0, the net positive charge of the lysozyme molecule is so large that the lysozyme molecules are attached strongly on the gold nanoparticle. The surface of the gold nanoparticle partially oxidized as -O-20, 21 binds as well as the positively charged amino groups (-NH3+) of lysozyme as monolayer.29 However, the lysozyme aggregation does not occur because of a repulsion force exerted between the lysozyme molecule on the gold nanoparticle and another incoming one. It follows that the lysozyme molecules are not efficiently degraded. The BSA molecules do not interact with the surface of the gold nanoparticle in this pH range and therefore are not degraded, because the net charge of the BSA molecule is almost zero. In a pH of ∼4.0, the lysozyme molecule is negatively charged so highly that it is not efficiently degraded as described in the case of the pH of 5.0–6.0. On the other hand, the net charge of the BSA molecules is positive, so that the BSA molecules tend to aggregate with other incoming BSA molecules. Namely, the BSA molecules are selectively degraded. The maximum value
J. Phys. Chem. C, Vol. 113, No. 13, 2009 5029 of the degradation rate of BSA at the pH of ∼4.0 is much smaller than that of lysozyme at the pH of ∼9.0 because the absolute value of the ζ-potential of the gold nanoparticle at the pH of ∼4.0 is smaller than that at the pH of ∼9.0, and therefore the interaction of the gold nanoparticle with the BSA molecules at the pH of ∼4.0 is also smaller than that with the lysozyme molecules at the pH of ∼9.0. In a pH range below 3.0, the absolute value of the ζ-potential of the gold nanoparticle decreases steeply with decrease of the pH of the solution, and accordingly the lysozyme and BSA molecules are not attached well to the gold nanoparticle. The net charges of the lysozyme and BSA molecules are so large that they do not aggregate in the vicinity of the gold nanoparticle due to a repulsion force. Therefore, the degradation rates of lysozyme and BSA decrease. The width and the height of the surface plasmon band of gold nanoparticles at ∼525 nm increased and decreased with laser irradiation, respectively, as shown in Figure 3. This finding shows that the radius of the gold nanoparticle decreases with laser irradiation. The absolute value of the ζ-potential of the gold nanoparticle conjugated with BSA or lysozyme molecules is smaller than that in a solution containing the gold nanoparticle alone because the ζ-potential of the gold nanoparticle alone and the net charge of the BSA or lysozyme molecules are opposite.30 Therefore, the nanoparticle conjugated with the BSA or lysozyme molecules has a tendency to aggregate. However, a small red shift (10 nm) of the plasmon band of the gold nanoparticle was observed for the solution containing BSA and lysozyme molecule as compared with that in the pure water solution. This indicates that the contact of the surface of the gold nanoparticles is prevented by attached BSA or lysozyme molecules. The shift arises from the presence of the layer of BSA or lysozyme molecules attached to the surface of the gold nanoparticle, which causes the refractive index of the surrounding of the gold nanoparticles to change.31 On the other hand, a large 40 nm red shift of the absorption wavelength of the plasmon band of the gold nanoparticle at the pH of 4.0 was observed after laser irradiation. This indicates that the exposed surfaces of the gold nanoparticles interact with each other due to disappearance of the layer of BSA. The solution contains two different proteins, BSA and lysozyme in the present study, while the solution contains lysozyme alone in the previous study.15 Nevertheless, the degradation rates 1/τ for lysozyme at the pH of 11.0 was larger than that at the pH of 4.9 in both the cases. This supports that the mechanism of the degradation proposed in both the studies is correct. These findings lead us to conclude that one can degrade proteins selectively in an aqueous solution containing gold nanoparticles by irradiation of a pulsed laser by choosing an appropriate value of the pH. An advantage of this method is that the protein degradation can be performed in a selective manner by controlling the protein-gold nanoparticle interaction and the protein-protein interaction by changing the pH of the solution. Acknowledgment. This research was supported by the Special Cluster Research Project of Genesis Research Institute, Inc. References and Notes (1) Grua, P.; Morreeuw, J. P.; Bercegol, H.; Jonusauskas, G.; Vallée, F. Phys. ReV. B 2003, 68, 035424. (2) Eah, S. -K.; Jaeger, H. M.; Scherer, N. F.; Lin, X. -M.; Wiederrecht, G. P. Chem. Phys. Lett. 2004, 386, 390. (3) Link, S.; El-Sayed, M. A. ReV. Phys. Chem. 2000, 19, 409. (4) Takeda, Y.; Kondow, T.; Mafuné, F. Nucleosides, Nucleotides Nucleic Acids 2005, 24, 1215.
5030 J. Phys. Chem. C, Vol. 113, No. 13, 2009 (5) Hüttmann, G.; Radt, B.; Serbin, J.; Birngruber, R. Proc. SPIE 2003, 5142, 88. (6) Hüttmann, G.; Birngruber, R. IEEE J. Quantum Electron. 1999, 5, 954. (7) Strauss, M.; Amendt, P. A.; London, R. A.; Maitland, D. J.; Glinsky, M. E.; Lin, C. P.; Kelly, M. W. Proc. SPIE 1997, 2975, 261. (8) Volkov, A. N.; Sevilla, C.; Zhigilei, L. V. Appl. Surf. Sci. 2007, 253, 6394. (9) O’Neal, D. P.; Hirsch, L. R.; Halas, N. J.; Payne, J. D.; West, J. L. Cancer Lett. 2004, 209, 171. (10) Logothetidis, S. Hippokratia 2006, 10, 7. (11) Letfullin, R. R.; Joenathan, C.; George, T. F.; Zharov, V. P. Nanomedicine 2006, 1, 473. (12) Pitsillides, C. M.; Joe, E. K.; Wei, X.; Anderson, R. R.; Lin, C. P. Biophys. J. 2003, 84, 4023. (13) Zharov, V. P.; Mercer, K. E.; Galitovskaya, E. N.; Smeltzery, M. S. Biophys. J. 2006, 90, 619. (14) Mafune´, F.; Kohno, J.; Takeda, Y.; Kondow, T.; Sawabe, H. J. Phys. Chem. B 2001, 105, 5114. (15) Takeda, Y.; Kondow, T.; Mafune´, F. J. Phys. Chem. B 2006, 110, 2393. (16) Mafune´, F.; Kohno, J.; Takeda, Y.; Kondow, T. J. Phys. Chem. B 2002, 106, 8555. (17) The solution was placed in an optical cell equipped with a stirrer at the bottom of the cell. An output of the second harmonic of the Nd: YAG laser (pulse duration, 10 ns) at a wavelength of 532 nm, which is close to that of the surface plasmon band of gold nanoparticles, was focused onto an area of 1.8 mm2 in the solution by a lens having a focal length of 250 mm. The amounts of the proteins in the solution were determined by
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