pubs.acs.org/Langmuir © 2009 American Chemical Society
Quantitative Studies of the Interactions of Metalloproteins with Gold Nanoparticles: Identification of Dominant Properties of the Protein that Underlies the Spectral Changes Elizabeth A. M. Lunt, Mark C. Pitter, and Paul O’Shea* Cell Biophysics Group, School of Biology & Institute of Biophysics, Imaging and Optical Science, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom Received April 1, 2009. Revised Manuscript Received June 13, 2009 The interaction of cytochrome C and a number of its components such as the apo protein, heme and a coordinated iron with gold nanospheres, has been investigated. The role of the heme group and its effect on the observed spectroscopic properties following binding of cytochrome C to the gold surface has been evaluated. Binding of the heme group directly to the gold is not observed, but the presence of the heme group and its effect on the interaction with the metal surface is shown to be influential. Other factors such as the metal oxidation state and the metal-free heme are also studied. A comparison to serum albumin binding as a nonmetallic protein provides further insight into the interaction characteristics.
Introduction Cytochrome C (cyt C) is a small (12 kDa), heme containing protein. It functions as a mobile carrier of single electrons and is also known to be involved in additional cellular processes associated with programmed cell death (apoptosis).1 The structure of cyt C is well characterized as is the relation of this to its function in electron transport, particularly as an electron carrier noncovalently attached to the outer surface of the inner mitochondrial membrane.2 These properties have made it a target for a range of studies to explore the interactions of proteins with surfaces and particularly with electrodes and noble metals. The electrochemistry of cyt C upon interaction with gold surfaces has been explored extensively.3 It is known, however, that cyt c may become partially denatured on some electrode surfaces, leading to deactivation. To overcome this, a negatively charged self-assembled monolayer (SAM) on the metal surface has been utilized to which cyt C will bind facilitating electrochemical studies.4-6 In other work, cyt C was attached covalently to SAMs for electrochemical and orientational studies using different analytical approaches.3,7,8 Nanoparticles provide a convenient means to study protein-gold interactions, and over the past few years these have proved to offer a versatile platform.9-11 Their optical properties permit study of these interactions using an array of *Corresponding author. E-mail:
[email protected].
(1) Liu, X.; Kim, C.; Yang, J.; Jemmerson, R; Wang, X. Cell 1996, 86, 147. (2) Skulachev, V. P. FEBS Lett. 1998, 423, 275. (3) Holt, K. B. Langmuir 2006, 22, 4928. (4) de Groot, M. T.; Evers, T. H.; Merkx, M.; Koper, M. T. M. Langmuir 2007, 23, 729. (5) Fedurco, M. Coord. Chem. Rev. 2000, 209, 263. (6) Eddowes, M. J.; Hill, H. A. O. J. Am. Chem. Soc. 1979, 101, 4461. (7) Nakano, K.; Yoshitake, T.; Yamashita, Y.; Bowden, E. F. Langmuir 2007, 23, 6270. (8) Sagara, T.; Kubo, T.; Hiraishi, K. J. Phys. Chem. B 2006, 110, 16550. (9) Taton, T. A.; Mirkin, C. A.; Letsinder, K. L. Science 2000, 289, 1757. (10) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (11) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607. (12) Jain, P. K.; Kyoeng, S. L.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 7238.
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techniques12 including absorption,13 SPR,14,15 SERS,16 and light scattering.17,18 Similarly, nanoparticle conjugates to biomacromolecules have been used in several applications including electron microscopy,19 immunoassays,20 and biosensors.21,22 Gold nanoparticles have been utilized in a variety of studies with cyt C including electrochemical, structural, and conformational approaches. Proteins bound to gold nanoparticles have been shown to retain their biological activity.18,23-25 Cyt C has been shown to stabilize gold nanoparticles in electrolytes at high pH by the formation of bioconjugated particles. These have been used to study the stability and control of orientation and the effect of particle size on conformational change.26-28 SAM coated particles have been used to facilitate further studies on conformational changes induced by pH29 and the (13) Englebienne, P.; Van Hoonacker, A.; Valsamis, J. Clin. Chem. 2000, 46, 2000. (14) Roll, D.; Malicka, J.; Gryczynski, I.; Gryczynski, Z.; Lakowicz, J. R. Anal. Chem. 2003, 75, 3440. (15) Englebienne, P.; Van Hoonacker, A.; Verhas, M.; Khlebtsov, N. G. Comb. Chem. High Throughput Screening 2003, 6, 777. (16) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. J. Phys.: Condens. Matter 2002, 14, R597. (17) Khlebtsov, N. G.; Bogatyrev, V. A.; Melinkov, A. G.; Dykman, L. A.; Khlebtsov, B. N.; Krasnov, Ya. M. J. Quant. Spectrosc. Radiat. Transfer 2004, 89, 133. (18) Lunt, E. A. M.; Pitter, M. C.; Somekh, M. G.; O’Shea, P. J. Nanosci. Nanotechnol. 2008, 8, 4335. (19) Hayat, M. A. Ed. Colloidal Gold: Principles, Methods and Applications; Academic: San Diego, 1989, Vols. 1, 2; 1991, Vol. 3. (20) Hirsch, L. R.; Jackson, J. B.; Lee, A.; Halas, N. J.; West, J. Anal. Chem. 2003, 75, 2377. (21) Bao, P.; Frutos, A. G.; Greef, C.; Lahiri, J.; Muller, U.; Peterson, T. C.; Warden, L.; Xie, X. Anal. Chem. 2002, 74, 1792. (22) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884. (23) Tom, R. T.; Samal, A. K.; Sreeprasad, T. S.; Pradeep, T. Langmuir 2007, 23, 1320. (24) Chah, S.; Hammond, M.; Zare, R. Chem. Biol. 2005, 12, 323–328. (25) Wang, L.; Wang, E. Electrochem. Commun. 2004, 6, 49–54. (26) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404. (27) Jiang, X.; Lei, Z.; Jiang, J.; Qu, X.; Wang, E.; Dong, S. ChemPhysChem 2005, 6, 1613. (28) Jiang, X.; Jiang, J.; Jin, Y.; Wang, E.; Dong, S. Biomacromolecules 2005, 6, 46. (29) Chah, S.; Hammond, M. R.; Zare, R. N. Chem. Biol. 2005, 12, 323.
Published on Web 07/14/2009
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effects of variation in the SAM headgroup on protein orientation and electrochemical behavior.30-32 A wide variety of methods have been utilized during these studies including circular dichroism, UV-vis spectroscopy, infrared spectroscopy,28 cyclic voltammetry,33 surface enhanced Raman scattering,26 mass spectrometry,30 and surface plasmon resonance.29 Of particular relevance to the present study are the spectroscopic changes occurring during the course of the nanoparticle-protein interaction that appear to be dependent on the nature of the medium and on the size of the nanoparticles.28,29 In an effort to gain further insight into the interactions of cytochrome C and nanoparticles, a comprehensive study of the effects of cyt C and several of its close derivatives on 100 nm gold particles using UV-vis spectrometry has been carried out. The interactions are monitored over time, and the change in the spectrum of the gold nanoparticles is explored. Particular focus is directed toward the interaction of heme with the gold surface. The effect of oxidation state, heme removal, and the use of cyt C from different origins are also evaluated.
Experimental Section Materials. Gold colloid (100 nm) was obtained from British Biocell International (UK). Bovine cytochrome C, hemin, protoporphryin IX, potassium ferricyanide, HEPES, and bovine serum albumin were obtained from Sigma Aldrich. Human cytochrome C and recombinant human apocytochrome C were obtained from R&D Systems. UV-vis spectra were collected on a Biochrom Libra S32 PC spectrometer at 25 °C using a far-UV quartz cuvette. Background scans of 10 mM HEPES buffer were taken for each experiment. Nanoparticles were dialyzed overnight into 10 mM HEPES buffer before use. Solutions were stored at 4 °C, excluded from light, except for cyt C proteins, which were stored in aliquots at -20 °C and defrosted to 4 °C as required (i.e., the protein was subjected to only one freeze-thaw cycle prior to study after which it was discarded). Studies with Bovine Cytochrome C. Gold colloid (0.5 mL, 100 nm) in 10 mM HEPES buffer pH 7.4 was diluted with HEPES buffer to give a final volume of 2 mL following the protein addition. Absorption scans were taken every 10 min for 2 h (200-900 nm). Cyt C (0.5 mM) in HEPES buffer was added after the initial scan to give a final concentration of 5 μM. Control scans were carried out of corresponding quantities of cyt C in HEPES buffer over 2 h and gold colloid after the addition of HEPES buffer (all additions were made after the initial scan). These studies were repeated with the addition of 0.5 mM cyt C to give a final concentration of 2.5 μM. For scans of reduced cyt C, 20 μL of a 0.5 mM cyt C solution was mixed with 10 μL of a 1 M ascorbic acid solution in HEPES buffer, pH 7.4, and incubated in the dark at room temperature for 2 h. Gold colloid (0.5 mL, 100 nm) was diluted with 1.47 mL of buffer. Absorption scans and appropriate controls were obtained as for cyt C. Further control experiments were carried out to study the effects of ascorbic acid as a gentle reductant on gold particles at the same final concentration. Cyt C was prepared in phosphate buffered saline (PBS) pH 7.4 at 0.5 mg/mL. This solution was added to after the initial scan to give a final concentration of 5 μM in a total volume of 2 mL. Control scans were carried out of cyt C in buffer and gold colloid after the addition of PBS (all additions were made after the initial (30) Bayraktar, H.; You, C.; Rotello, V. M.; Knapp, M. J. J. Am. Chem. Soc. 2007, 129, 2732. (31) Aubin-Tam, M.; Hamad-Schifferli, K. Langmuir 2005, 21, 12080. (32) Kaufman, E. D.; Belyea, J.; Johnson, M. C.; Nicholson, Z. M.; Ricks, J. L.; Shah, P. K.; Bayless, M.; Pettersson, T.; Feldoto, Z.; Blomberg, E.; Claesson, P.; Franzen, S. Langmuir 2007, 23, 6053. (33) Jiang, X.; Shang, L.; Wang, Y.; Dons, S. Biomacromolecules 2005, 6, 3030.
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scan). Studies were repeated with 0.5 mg/mL cyt C in PBS to give a final concentration of 2.5 μM. Studies with Bovine Serum Albumin. BSA (0.5 mM) in 10 mM HEPES, pH 7.4, was added to the gold colloid suspension after the initial scan to give a final concentration of 5 μM. Scans were taken every 10 min for 1 h. Appropriate control scans of BSA and gold nanoparticles were obtained. BSA (0.5 mg/mL) in PBS pH 7.4 was added to the gold colloid suspension in the same way to give a final concentration of 5 μM. Scans were taken every 10 min for 2 h. Appropriate control scans of BSA and gold colloid were obtained.
Studies with Potassium Hexacyanoferrate (Ferri/Ferro Cyanide). Potassium ferricyanide solution (0.1 M) in 10 mM HEPES pH 7.4 was added to the gold colloid suspension after the initial scan to give a final concentration 0.5 mM. Scans were taken every 10 min for 1 h. Appropriate control scans of potassium ferricyanide and gold colloid were obtained. Identical studies were performed with ferrocyanide (in the presence of cytochrome c, this would lead to reduction) and thus facilitating clear identification of any effect of the oxidation state of the Fe center) Studies with Hemin and Protoporphryin IX. One mM solutions in 5% DMSO in 10 mM HEPES pH 7.4 of both compounds were used. Solvents were flushed with nitrogen before use, and solutions were maintained under nitrogen in the dark at 4 °C. The appropriate solution was added to the gold colloid after the initial scan to give a final concentration of 5 μM. Scans were taken every 10 min for 1 h. Control scans were obtained of the appropriate compound and of gold colloid. Studies with Apocytochrome C. Recombinant human apocytochrome C (apocyt C) was obtained as a 0.5 mg/mL solution in phosphate buffered saline (PBS). Experiments were carried out in a 250 μL cuvette, and volumes were scaled accordingly to maintain the same final concentrations. Apocyt C solution was added to the gold colloid after the initial scan. Scans were taken every 10 min for 2 h. Further experiments were carried out at the same concentration over a 1 h period with scans every 3 min. Appropriate control scans of apocyt C and gold colloid were obtained. Studies with Human Cytochrome C. Human cytochrome C was dissolved in PBS, pH 7.4, at 0.5 mg/mL. Experiments were carried out in a 75 μL cuvette, and volumes were scaled appropriately. Human cyt C solution was added after the initial scan to give a final concentration of 2 μM, and the experiment run for 2 h. Appropriate control scans of human cyt C and gold colloid were obtained. Data Analysis. Experiment scans were normalized using compound controls. The human cyt C scans and controls were zeroed before normalization. Changes in peak maximum were determined using an in-house polynomial curve fit (MATLAB). The analytical protocol defining wavelength changes is outlined in ref 18.
Results A number of studies were carried out to define the interaction of cyt C with 100 nm gold nanospheres. The results of the binding of 5 μM bovine cyt C to nanospheres on the UV-vis absorption spectrum of the nanoparticle during a 2 h period are shown (Figure 1A). It is clear that a significant broadening of the spectrum curve was observed, combined with a decrease in the absorption. After 2 h, the spectrum for the 100 nm gold nanospheres is largely flat. No significant peaks are observed at longer wavelengths although other broad features are discernible. Kinetic analysis of the data (shown as the fitted solid line) indicates a pseudo-first-order reaction with an overall change in maximum peak wavelength of 57 nm. Studies carried out using half the initial concentration (2.5 μM) of bovine cyt C indicated a concentration dependence for the rate of binding and the total shift in the peak wavelength (Figure 1B). DOI: 10.1021/la901148q
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Figure 1. Interaction of cyt C in HEPES with gold nanoparticles. (A) Sample experimental data showing the peak shift and broadening during the course of an experiment (bovine cyt C 5 μM). (B) Change in maximum peak wavelength of the gold nanospheres in the presence of bovine cyt C at 5 μM. Kinetics: y=a exp(-bx) þ c; max dev: 6.66, R2=0.956, a = -56.6 ((2.06), b=0.0513 ((0.00423), c=56.6 ((0.920). (C) Change in maximum peak wavelength of the gold nanospheres in the presence of bovine cyt C at 2.5 μM. Kinetics: y=a exp(-bx) þ c; max dev: 12.7, R2 =0.842, a=-52.8 ((3.89), b=0.0543 ((0.00901), c=52.8 ((1.67). (D) Change in maximum peak wavelength of the gold nanospheres in the presence of reduced bovine cyt C at 5 μM. Kinetics: y=a exp(-bx) þ c; max dev: 5.45, R2=0.957, a=-40.0 ((1.43), b= 0.0286 ((0.00283), c=39.6 ((1.15). Table 1. Summary of the Rate Constants for the Interaction of Various Proteins with 100 nm Gold Nanoparticles (*Estimated Value) protein system
concentration (μM)
Rate constant (mol L-1 s-1)
ionic strength
bovine cyt C HEPES bovine cyt C HEPES reduced bovine cyt C HEPES bovine cyt C PBS bovine cyt C PBS human cyt C PBS apo cyt C PBS
5 2.5 5 5 2.5 2.5 5
0.0513 0.0286 0.0543 0.0322 0.0318 0.00932 0.266 0.00588
0.01 0.01 0.01 0.0267 0.0183 0.0183 0.0687*
The ratio of the reaction rates is 0.56, indicating a much reduced rate with the overall change in peak wavelength of 38.5 nm. The rate constants for these and the further studies described below are collated in Table 1. It was considered important to study further the underlying basis of these dramatic spectral observations accompanying the metal-protein interaction. Particular attention was paid to the property of cyt C associated with the delivery of electrons as part of its normal biological function as it possesses an iron-based metal center that undergoes a simple 1-electron oxidationreduction reaction with the other components of the mitochondrial cytochrome system as well as several others in (e.g.) plants etc. This resides within the heme-porphyrin system. Thus, it was of interest to identify whether this structure was the molecular moiety that underlies the spectral changes of the nanoparticles or whether it was more a property of the broader protein structure that envelopes the metal center within the protein. Exploration of the effects of the oxidation state of the bovine cyt C on the 10102 DOI: 10.1021/la901148q
interaction with gold nanospheres was therefore deemed important and similar experiments (Figure 1C,D) were carried out with bovine cyt C, reduced using ascorbate immediately prior to the addition to the nanoparticles. The same broadening of the spectrum over the 2 h period was observed, and kinetic analysis yielded a very similar rate constant and an overall peak wavelength change of 53 nm. Control studies were carried out to determine the effect of the excess ascorbate on the absorption spectra of gold nanospheres resulted in essentially insignificant changes in maximum peak wavelength of
