2714
Langmuir 2007, 23, 2714-2721
pH-Dependent Protein Conformational Changes in Albumin:Gold Nanoparticle Bioconjugates: A Spectroscopic Study Li Shang, Yizhe Wang, Junguang Jiang, and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China ReceiVed July 17, 2006. In Final Form: October 6, 2006 The conformational changes of bovine serum albumin (BSA) in the albumin:gold nanoparticle bioconjugates were investigated in detail by various spectroscopic techniques including UV-vis absorption, fluorescence, circular dichroism, and Fourier transform infrared spectroscopies. Our studies suggested that albumin in the bioconjugates that was prepared by the common adsorption method underwent substantial conformational changes at both secondary and tertiary structure levels. BSA was found to adopt a more flexible conformational state on the boundary surface of gold nanoparticles as a result of the conformational changes in the bioconjugates. The conformational changes at pH 3.8, 7.0, and 9.0, which corresponded to different isomeric forms of albumin, were investigated, respectively, to probe the pH effect on the conformational changes of BSA in the bioconjugates. The results showed that the pH of the medium influenced the changes greatly and that fluorescence and circular dichroism studies further indicated that the changes were larger at higher pH.
Introduction The burgeoning field of nanotechnology promises to revolutionize many scientific fields. For instance, the integration of nanotechnology with biology is expected to produce major advances in molecular diagnostics, material science, and bioengineering.1-4 Within these general activities, the use of protein:nanoparticle conjugates for applications in sensing, assembly, imaging, and control has substantially advanced.5-8 The conjugation of protein with nanoparticles not only affords stabilization to the system, but more importantly, it also introduces biocompatible functionalities into these nanoparticles for further biological interactions or coupling.3,9 However, in most cases, the protein undergoes more or less structural changes at the boundary surface of nanoparticles in the conjugates.10-15 The resulting changes in structure and function actually can have * To whom correspondence should be addressed. Fax: 86-431-5689711. E-mail:
[email protected]. (1) Niemeter, C. M. Angew. Chem., Intl. Ed. 2001, 40, 4128-4158. (2) Maxwell, D. J.; Taylor, J. R.; Nie, S. M. J. Am. Chem. Soc. 2002, 124, 9606-9612. (3) Katz, E.; Willer, I. Chem. Phys. Chem. 2004, 5, 1084-1104. (4) You, C. C.; Verma, A.; Rotello, V. M. Soft Matter 2006, 2, 190-204. (5) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404-9413. (6) Gole, A.; Dash, C.; Soman, C.; Sainkar, S. R.; Rao, M.; Sastry, M. Bioconjugate Chem. 2001, 12, 684-690. (7) Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.; Franzen, S.; Feldheim, D. L. J. Am. Chem. Soc. 2003, 125, 4700-4701. (8) Mason, J. N.; Farmer, H.; Tomlinson, I. D.; Schwartz, J. W.; Savchenko, V. Defelice, L. J.; Rosenthal, S. J.; Blakely, R. D. J. Neurosci. Methods 2005, 143, 3-25. (9) Pasquasto, L.; Pengo, P.; Scrimin, P. Biological and Biomimetic Applications of Nanoparticles. In Nanoparticles: Building Blocks for Nanotechnology; Rotellom, V., Ed.; Kluwer Academic/Plenum: New York, 2004; pp 251-282. (10) Zhou, H. S.; Aoki, S.; Honma, I.; Hirasawa, M.; Nagamune, T.; Komiyama, H. Chem. Commun. 1997, 605-606. (11) Lundqvist, M.; Sethson, I.; Jonsson, B. H. Langmuir 2004, 20, 1063910647. (12) Peng, A. G.; Hidajat, K.; Uddin, M. S. Colloids Surf., B 2004, 2, 457461. (13) Jiang, X.; Jiang, J.; Jin, Y.; Wang, E.; Dong, S. Biomacromolecules 2005, 6, 46-53. (14) Aubin-Tam, M. E.; Hamad-Schifferli, K. Langmuir 2005, 21, 1208012084. (15) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2006, 128, 39393945.
profound effects in the related applications of the bioconjugates since the conformational changes of the conjugated protein may possibly mean the loss of biological activity or the activation of immune response.16-17 Moreover, detailed mapping of protein conformational changes can help identify optimal conditions to preserve functionality following the conjugation and direct the further applications. Thus, a fundamental understanding of the conformational behavior of proteins in protein:nanoparticle conjugates is of critical importance in developing the bioconjugated nanomaterials. A variety of methods has been developed to characterize the protein conformational changes, and one of the most commonly adopted methods is the spectroscopic method, including circular dichroism (CD),18-19 Fourier transform infrared (FTIR) spectroscopy,20-22 fluorescence spectroscopy,23-24 and so on. In the practical study, a combination of different techniques is often necessary if one wants to obtain a comprehensive understanding of the protein conformational behavior. In the study presented here, the albumin:nanoparticle bioconjugate was chosen as the model system. Bovine serum albumin (BSA), whose structure and property are well-characterized, is a convenient model for the fundamental studies of protein: nanoparticle conjugates.25-27 Moreover, BSA can undergo structural changes very easily and hence can be used as a good (16) Baron, M. H.; Revault, M.; Servagent-Noinville, S.; Abadie, J.; Quiquampoix, H. J. Colloid Interface Sci. 1999, 214, 319-332. (17) Brandes, N.; Welzel, P. B.; Werner, C.; Kroh, L. W. J. Colloid Interface Sci. 2006, 299, 56-59. (18) Greenfield, N. J. Trends Anal. Chem. 1999, 18, 236-244. (19) Kelly, S. M.; Jess, T. J.; Price, N. C. Biochim. Biophys. Acta 2005, 1751, 119-139. (20) Haris, P. I.; Chapman, D. Trends Biol. Sci. 1992, 17, 328-333. (21) Chittur, K. K. Biomaterials 1998, 19, 357-369. (22) Zhang, J.; Yan, Y. B. Anal. Biochem. 2005, 340, 89-98. (23) Royer, C. A. Chem. ReV. 2006, 106, 1769-1784. (24) Matyus, L.; Szollosi, J.; Jenei, A. J. Photochem. Photobiol., B 2006, 83, 223-236. (25) Carter, D. C.; Ho, X. J. AdV. Protein Chem. 1994, 45, 153-203. (26) Kaibara, K.; Okazaki, T.; Bohidar, H. B.; Dubin, P. L. Biomacromolecules 2000, 1, 100-107. (27) Papadopoulou, A.; Green, R. J.; Frazier, R. A. J. Agric. Food Chem. 2005, 53, 158-163.
10.1021/la062064e CCC: $37.00 © 2007 American Chemical Society Published on Web 01/24/2007
Changes in Albumin:Gold Nanoparticle Bioconjugates
model for conformational change studies.15,28 Recently, several studies on the conjugation of BSA:nanoparticle conjugates have been reported. For example, Jose-Yacaman et al. reported the synthesis of gold nanoparticles directly conjugated to BSA by chemical reduction in aqueous solution.29 In another study, highly ordered silver sulfide nanorods conjugated with BSA have been successfully achieved in the lab of Ren.30 Using ξ-potential and quartz crystal microbalance measurements, Franzen et al. showed that the conjugation of BSA with gold nanoparticles occurred mostly by an electrostatic mechanism.31 Despite wide interest in the albumin:nanoparticle bioconjugates, the conformational behavior of BSA on conjugation with nanoparticles, which is of great importance in the application of these bioconjugated nanomaterials, is still unclear at present. With this in mind, we then performed our studies on the protein conformational changes in the BSA:gold nanoparticle conjugates by various spectroscopic methods including UV-vis, fluorescence, CD, and FT--IR techniques. Gold nanoparticles were adopted because they are the most commonly used nanoparticles in the bioconjugation study.6,10,13-14 The method adopted in preparing the bioconjugates in this study was attaching the protein to the nanoparticles through adsorption despite many other methods existing because this simple method has been extensively adopted in many different protein:nanoparticle conjugates,5,32-33 which made the study here representative and comparable. Particularly, we investigated the pH effect on the conformational changes of BSA in the bioconjugates since albumin is known to undergo reversible conformational isomerization as a consequence of pH changes.28,34 The pH-dependent forms of albumin are classified as N, for normal or native forms, which is predominant at neutral pH; B, for the basic form occurring above pH 8; F, for the fast migrating form produced abruptly at pH values less than 4.3; and E, for the expanded form at pH less than 3.5. All these forms display characteristic structure and functions, and the conformational transition between different forms has physiological significance. Therefore, four pH values, 2.7 (E form), 3.8 (F form), 7.0 (N form), and 9.0 (B form), were chosen in the present study to investigate the pH effect on the conformational changes of BSA in the albumin:gold nanoparticle bioconjugates. Experimental Procedures Materials. Bovine serum albumin (BSA, fraction V), purchased from Sigma , was used without further purification. The stock solution of BSA was prepared in 0.01 M phosphate buffer solution (PBS) of pH 7.0, containing 0.01 M NaCl. PBS with other pH values used in the experiment was prepared by adding concentrated HCl or NaOH to the PBS of pH 7.0. The concentration of BSA was measured spectrophotometrically using a molar absorptivity of 44 000 mol-1 cm-1 at 278 nm. The HAlCl4‚3H2O and trisodium citrate were purchased from Beijing Chemical Company. All other reagents were of analytical reagent grade and used as received. The water used was purified through a Millipore system. Preparation of Gold Nanoparticles (GNP). All glassware used in the experiment was cleaned in a bath of freshly prepared 3:1 (28) Giacomelli, C. E.; Avena, M. J.; De Pauli, C. P. J. Colloid Interface Sci. 1997, 188, 387-395. (29) Burt, J. L.; Gutierrez-Wing, C.; Miki-Yoshida, M.; Jose-Yacaman, M. Langmuir 2004, 20, 11778-11783. (30) Yang, L.; Xing, R.; Shen, Q.; Jiang, K.; Ye, F.; Wang, J.; Ren, Q. J. Phys. Chem. B. 2006, 110, 10534-10539. (31) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21, 9303-9307. (32) Baudhuin, P.; van der Smissen, P.; Beauvois, S.; Courtoy, P. J. Colloidal Gold: Principles, Methods, and Applications; Academic Press: New York, 1989. (33) Shenton, W.; Davis, S. A.; Mann, S. AdV. Mater. 1999, 11, 449-452. (34) Ahmad, B.; Parveen, S.; Khan, R. H. Biomacromolecules 2006, 7, 13501356.
Langmuir, Vol. 23, No. 5, 2007 2715 HCl/HNO3 and rinsed thoroughly in water prior to use. Gold nanoparticles were prepared following Frens’ method.35 Typically, a 50 mL aqueous solution of HAuCl14 (1 mM) was heated to boiling, then 5 mL of trisodium citrate (1%) was added. The boiling solution was stirred for another 30 min and then stored at 4 °C prior to use. The size and morphology of GNP were characterized with TEM (JEOL 2010) and UV-vis spectroscopy (Cary 500). Preparation of BSA:Gold Nanoparticle Conjugates. The test solutions of the bioconjugates were prepared by mixing BSA and GNP in the PBS of different pH values and then incubated at 4 °C for at least 30 min before the spectra were obtained. A series of the bioconjugates with different molar ratios of BSA/GNP were prepared by keeping the concentration of BSA constant while varing the concentration of GNP. To retain their original conformational states in the solution, and also to prevent shear forces from disrupting the structure of the bioconjugates, no separation techniques (i.e., centrifugation or filitration) were employed in the experiment. All the measurements in the following discussion were performed at room temperature (293 K). Fluorescence Measurements. Fluorescence measurements were performed on a LS-55 Luminescence Spectrometer (Perkin-Elmer). The spectra were recorded in the wavelength range of 310-500 nm upon excitation at 295 nm, using 10 nm/10 nm slit widths, and each spectrum was the average of three scans. An excitation wavelength of 305 nm was adopted when investigating the red edge excitation shift (REES) effect of the albumin. A 1.00 cm path length rectangular quartz cell was used for these studies. To avoid self-absorption and inner filter effects,36 very dilute solutions (BSA concentration of 1 × 10-6 M and GNP concentration in the range of 2.0 × 10-11 to 3.3 × 10-10 M) were used in the experiment. Also, appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background. The experiments were repeated and found to be reproducible within experimental errors. Circular Dichroism (CD) Measurements. CD measurements were made on a 62A DS CD spectrometer (AVIV) with a 1.0 cm path length rectangular quartz cell controlled by a thermoelectric cell holder (AVIV). CD spectra were taken in a wavelength range of 200-250 nm, and each spectrum was the average of three scans. The results were expressed as mean residue ellipticity (MRE) in deg cm2 dmol-1.34 The value of MRE can be obtained using the equation MRE ) [θ]/(10nlC), where [θ] is the CD in millidegrees obtained from the spectra, n is the number of amino acid residues (583 for BSA), l is the path length of the cell (1.0 cm), and C is the mol fraction of the protein. Then, the helical content of BSA can be calculated from the [θ] value at 208 nm according to the equation % helix ) {(-[θ]208 - 4000)/(33000 - 4000)}100 as described by Lu et al.37 The concentration of BSA in the CD study was 2.5 × 10-7 M, and the concentration of GNP in the bioconjugates was in the range of 2 × 10-10 to 1.1 × 10-9 M. Fourier Transform Infrared (FTIR) Measurements. FT-IR spectra were recorded on a Nicolet 520 FT-IR spectrometer (Nicolet) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector, and a KBr beam splitter. All spectra of the bioconjugates were taken via the ATR method with a resolution of 4 cm-1. A total of 128 interferograms were co-added in the experiment to ensure a good signal-to-noise ratio. Reference spectra (the exact solution just without protein) were recorded under identical conditions. The subtraction of the reference spectrum from the spectrum of the protein solution was carried out in accord with the criteria that a straight baseline was obtained between 2000 and 1750 cm-1.38 Second-derivative spectra were obtained with the derivative function of Ominic E.S.P. software. The concentrations of BSA and GNP in the bioconjugates in the FTIR study were 1.2 × 10-5 and 2.4 × 10-9 M, respectively. (35) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (36) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/Plenum Publishers: Dordrecht, The Netherlands, 2004. (37) Lu, Z. X.; Cui, T.; Shi, Q. L. Application of Circular Dichroism and Optical Rotatory Dispersion in Molecular Biology, 1st ed.; Science Press: Beijing, 1987. (38) Dong, A.; Huang, P.; Caughy, W. S. Biochemistry 1990, 29, 3303-3308.
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Figure 1. Absorption spectra of gold nanoparticles (curve a) and the albumin:gold nanoparticle conjugates at pH 2.7 (curve b), pH 3.8 (curve c), pH 7.0 (curve d), and pH 9.0 (curve e). Curve f is the absorption spectrum of BSA in 0.01 M PBS at pH 7.0.
Results and Discussion Characterization of GNP and BSA:GNP Conjugates. The as-prepared GNP solution exhibits a color of dark red, which is known to arise from the collective oscillation of the free conduction electrons induced by an interacting electromagnetic field.39 UV-vis absorption measurements (see curve a in Figure 1) indicated that the maximum wavelength of the surface plasmon resonance (SPR) was 522 nm. TEM results (not shown) showed that the GNPs were well-dispersed with an average size of 15 nm. The concentration of as-prepared GNP was then calculated to be approximately 10 nM, assuming that all gold in the HAuCl14 was reduced. Curves b-e in Figure 1 show the absorption spectra of the BSA:GNP bioconjugates at pH 2.7 (curve b), pH 3.8 (curve c), pH 7.0 (curve d), and pH 9.0 (curve e), while curve f shows the absorption spectrum of native BSA in PBS of pH 7.0. As shown, pure BSA exhibits an absorbance maximum at 278 nm, which mainly originates from aromatic residues and disulfide bonds in the protein. The position of this peak was found to remain almost unchanged after the conjugation at either pH, although the shape of the absorption band changed slightly. Moreover, due to the substantial contribution of GNP in this region, it was difficult to extract any valuable information relevant to the conformational behavior of BSA in the bioconjugates from the absorption spectra. In contrast, obvious changes of the SPR band were observed for the GNP after conjugating with the albumin at different pH. As compared with the absorbance of GNP in aqueous solution, the SPR band of GNP in the conjugates showed an apparent broadening and red shift, which indicated the formation of bioconjugates.5,40 The position of the maximum absorption was shifted from 522 to 524, 526, 531, and 532 nm for the bioconjugates at pH 9.0, 7.0, 3.8, and 2.7, respectively. It is noteworthy that at pH 2.7, a broad band due to the aggregation of the GNP at long wavelength (ca. 610 nm) could be observed, which indicated a poor stability of the bioconjugates prepared at pH 2.7. In fact, the conjugates prepared at pH 2.7 showed obvious precipitation after 1 week, while those prepared at other pH values were still steady within weeks. It is obvious that the albumin:GNP conjugates were more stable with increasing pH value. Because of the poor stability of the bioconjugates at pH 2.7, we will just discuss the results obtained at pH 3.8, 7.0, and 9.0 in the following sections. Also, it is necessary to note here that the amount of albumin in the conjugates prepared in the present study was all in excess than that needed for the saturated (39) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer: Berlin, 1995. (40) Chan, S.; Hammond, M. R.; Zare, R. N. Chem. Biol. 2005, 12, 323-328.
Shang et al.
Figure 2. Fluorescence emission spectra of 1 × 10-6 M BSA at pH 3.8 (curve a), pH 7.0 (curve b), and pH 9.0 (curve c) upon excitation at 295 nm in 0.01 M PBS.
coverage on the surface of gold nanoparticles.41 However, no efforts were made to remove the redundant proteins once considering that the free albumin molecules in the solution could offer more stabilization to the bioconjugates. On the other hand, any separation techniques that were employed on the adsorption prepared bioconjugates may disrupt their original structures and thus possibly affect the conformational behavior of protein in the conjugates. Fluorescence Study. Fluorescence spectroscopy is useful to obtain local information about the conformational and/or dynamic changes of protein. Typically, from the interpretation of fluorescence parameters, one can obtain information such as the degree of exposure of the fluorophore to the solvent and the extent of its local mobility.23 For proteins with intrinsic fluorescence, more specific local information can be obtained by selectively exciting the tryptophan (Trp) residues. BSA is known to possess two Trp residues.25 One of them is located on the bottom of hydrophobic pocket in domain II (Trp-213), whereas the other is on the surface of the molecule in domain I (Trp-134). Thus, on observing the fluorescence emission of Trp in the bioconjugates, information about the protein conformational behavior around the Trp residues can be obtained. Figure 2 shows the emission spectra of native BSA at different pH vlaues upon excitation at 295 nm. The choice of 295 nm as the excitation wavelength was to avoid the contribution from tyrosine residues. As shown, the fluorescence intensity was found to decrease with increasing the pH from 3.8 to 9.0, while the emission maximum shifted from 351 nm at pH 3.8 (curve a) to 352.5 nm at pH 7.0 (curve b) and 348.5 nm at pH 9.0 (curve c), respectively. These different fluorescent characteristics actually reflected different conformational states of BSA at pH 3.8, 7.0, and 9.0, which corresponded to the F, N, and B forms, respectively, as described before. Figure 3 shows the emission spectra of 1.0 × 10-6 M BSA in the albumin:GNP conjugates with increasing the concentration of GNP at pH 7.0. It could be seen from Figure 3 that the Trp fluorescence was quenched drastically upon conjugating with GNP. Moreover, with increasing the concentration of GNP in the bioconjugates, the fluorescence intensity decreased gradually accompanied by a slight blue shift of the emission maximum. Quenching of the fluorophore fluorescence by gold nanoparticles that results in energy transfer to metal particles has been reported earlier,42,43 and gold nanoparticles (41) Previous studies (i.e., Xie, H. et al. Anal. Chem. 2005, 75, 5797-5805) have reported that no more than 50 BSA molecules are adsorbed at monolayer coverage for the 15 nm citrate stabilized gold nanoparticles, while the molar ratio of BSA/GNP of the bioconjugates in the present study was no less than 200. (42) Dulkeith, E.; Morteani, A. C.; Niedereichholz, T.; Klar, T. A.; Feldmann, J.; Levi, S. A.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Moller, M.; Gittins, D. I. Phys. ReV. Lett. 2002, 89, 203002. (43) Ding, Y.; Zhang, X.; Liu, X.; Guo, R. Langmuir 2006, 22, 2292-2298.
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Langmuir, Vol. 23, No. 5, 2007 2717
Figure 3. Fluorescence emission spectra of 1 × 10-6 M BSA in the absence (curve a) and presence of GNP with concentrations in the range of 2.0 × 10-11 to 3.3 × 10-10 M (curves b-j) upon excitation at 295 nm at pH 7.0. Inset: Stern-Volmer plots of BSA with the increasing concentration of GNP.
are known to exhibit efficient energy transfer behavior as exited state quenchers. Thus, in the bioconjugates, where the protein is situated in the vicinity of gold nanoparticles, efficient energy transfer will occur between BSA and GNP. As a result, the emission of Trp residues in the albumin is quenched. Fluorescence intensity data were then analyzed using the Stern-Volmer equation36
F0 ) 1 + kqτ0[Q] ) 1 + KSV[Q] F where F0 and F are the maximum fluorescence intensities in the absence and presence of GNP, respectively, kq is the bimolecular quenching constant, τ0 is the lifetime of the fluorophore in the absence of quencher, KSV is the Stern-Volmer fluorescence quenching constant, which is a measure of the efficiency of quenching, and [Q] is the quencher concentration. The inset in Figure 3 shows the Stern-Volmer plots, F0/F versus [GNP], according to the previous equation. KSV, calculated by linear regression of the plots, was 2.7 × 109 M-1, which indicated a quite strong quenching ability of GNP.44 From the previous equation, we know that KSV ) kqτ0. For BSA, τ0 is known to be approximately 5 × 10-9 s;36 thus, kq ) 5.4 × 1017 M-1 s-1 was obtained. Since the maximum value of kq for a diffusion-controlled quenching process is about 1010 M-1 s-1, the higher value obtained here suggested that the quenching of Trp fluorescence occurred by a specific interaction between BSA and GNP.45,46 This also implied that the dominating quenching mechanism was static (formation of the bioconjugates).47 Furthermore, the blue shift of the emission maximum observed in the bioconjugates indicated the occurrence of conformational changes for BSA at tertiary structure levels since the shift in the position of emission maximum reflected the changes of the polarity around the Trp residues. Commonly, the red shift indicates that Trp residues are, on average, more exposed to the solvent, whereas the blue shift is a consequence of transferring Trp residues into a more hydrophobic environment.48 Thus, the blue shift here indicated that Trp residues were in a more hydrophobic environment due (44) Fan, C.; Wang, S.; Hong, J. W.; Bazan, G. C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 200, 6297-6301. (45) Ware, W. R. J. Phys. Chem. 1962, 66, 455-458. (46) Eftink, M. R. Fluorescence Quenching Reactions: Probing Biological Macromolecular Structures. In Biophysical and Biochemical Aspects of Fluorescence Spectroscopy; Dewey, T. G., Ed.; Plenum: New York, 1991. (47) Kang, J.; Liu, Y.; Xie, M.; Li, S.; Jiang, M.; Wang, Y. Biochim. Biophys. Acta 2004, 1674, 205-214. (48) Pan, B. F.; Gao, F.; Ao, L. M. J. Magn. Magn. Mater. 2005, 293, 252258.
Figure 4. Position of Trp emission maximum registered as a ratio of fluorescence intensities at two wavelengths: on the left (FL) and on the right (FR) slopes of the spectrum at pH 3.8 (A), pH 7.0 (B), and pH 9.0 (C).
to the tertiary structural change of the albumin when conjugated at the boundary surface of GNP. The Trp emission behavior in the bioconjugates at pH 3.8 and 9.0 was also investigated, and similar quenching of Trp fluorescence emission was observed in each case. Since the position of the emission maximum changed very slightly (not exceeding 5 nm), to evaluate the pH effect on the Trp emission more precisely, we then used a double wavelength method to evaluate the position of emission maximum as described by Bryszewska et al.49 Thus, we registered the ratio of fluorescence intensities at two wavelengths: on the left (FL) and right (FR) slopes of the spectrum. In our study, the wavelengths of FL and FR were both chosen to be 20 nm away from the emission maximum of native BSA at each pH (i.e., 332.5 and 372.5 nm for FL and FR, respectively, at pH 7.0). This method was proved to be sensitive to the shift of the emission maximum, thus providing a convenient avenue to investigate the pH effect on the Trp emission behavior. The results were listed in Figure 4. As shown, in all cases, the value of FL/FR increased gradually with increasing the concentration of GNP. A higher ratio of FL/FR actually meant a blue shift in the position of the emission maximum, which showed that BSA underwent similar tertiary structural changes at pH 3.8, 7.0, and 9.0 that the Trp residues were placed in a more hydrophobic environment as a result of the bioconjugation. The intrinsic reason of this change may lie in a more flexible conformation the albumin adopted on the surface of nanoparticles, which favored the proximity of Trp residues to the bulk surface of GNP. However, obvious differences could be observed from Figure 4 that the extent of changes varied at different pH values. As seen, the variation at pH 3.8 in Figure 4A was much smaller than that at pH 7.0 and 9.0 (Figure 4B,C), while the change at (49) Klajnert, B.; Stanislawska, L.; Bryszewska, M.; Palecz, B. Biochim. Biophys. Acta 2003, 1648, 115-126.
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Table 1. Red Edge Excitation Effects for Native BSA and That in Bioconjugates at Different pH Valuesa λem max (nm) pH
sample
λex: 295 nm
λex: 305 nm
∆λem max (nm)
3.8
BSA BSA:GNP BSA BSA:GNP BSA BSA:GNP
351 350 352.5 348 348.5 345.5
352.5 352 355 352 351.5 350.5
1.5 2 2.5 4 3 5
7.0 9.0
a The concentration of BSA and GNP in the BSA:GNP conjugates was 1 × 10-6 and 2.5 × 10-10 M, respectively.
pH 7.0 was a little smaller than that at pH 9.0. This was interesting since albumin at pH 7.0 and 9.0, which is larger than the isoeletric point of BSA (4.7), is negatively charged as a whole; thus, the electrostatic association of BSA with citrate-stabilized GNP was expected to be hampered. Previous studies suggested that BSA binding to citrate coated GNP mainly occurred by an electrostatic mechanism,31 so the conjugation of albumin with GNP should be weaker at pH 7.0 and 9.0. However, the larger change of Trp emission behavior here reflected a stronger interaction between them in both cases. This could then be rationalized by considering over 60 lysine residues placed on the surface of BSA, which could have electrostatic interactions with negative surfaces. Moreover, previous studies showed that besides electrostatic interactions, other types of forces such as hydrophobic interactions and coordination binding might also work in the conjugation of protein with nanoparticles.13,50 This pH-dependent change on the shift of the Trp emission maximum showed that the pH of the medium greatly influenced the conformational changes of protein in the bioconjugates at tertiary structure levels and that the change was larger with increasing the pH in the experimental range. The Trp emission in the bioconjugates was further investigated by red edge excitation shift (REES) experiments.51 REES is a shift in the emission maximum toward a higher wavelength caused by a shift in the excitation wavelength toward the red edge of the absorption band. The REES is due to the electronic coupling between Trp indole rings and neighboring dipoles and occurs when there are slow relaxations of solvent media. Thus, REES is particularly useful in monitoring motions around the Trp residues in the protein study.49 In our experiment, we chose to excite the Trp at both 295 and 305 nm to investigate the REES effect, and the results are listed in Table 1. The value of 4λem max is defined as the difference of the emission maximum between that excited at 295 nm and at 305 nm. As shown, native BSA showed a 1.5, 2.5, and 3 nm REES for pH 3.8, 7.0, and 9.0, respectively, indicating that Trp residues in the albumin were in a slight motionally restricted environment and that the extent of this restriction increased at higher pH. In the bioconjugates, the values all showed an increase (2, 4, and 5 nm for pH 3.8, 7.0, and 9.0, respectively). The increase of 4λem max meant that the introduction of GNP had an obvious impact on the mobility of the Trp microenvironment and that Trp residues faced more restrictions from their surroundings in the bioconjugates. Again, the extent of the increase in 4λem max after the conjugation followed an order of pH 9.0 (2 nm) > pH 7.0 (1.5 nm) > pH 3.8 (0.5 nm), which agreed with the result in the changes of the Trp emission maximum. The previous fluorescence study suggested that obvious changes of the albumin tertiary structure took place in the albumin:gold nanoparticle bioconjugates, which was reflected (50) Gao, D.; Tian, Y.; Bi, S.; Chen, Y.; Yu, A.; Zhang, H. Spectrochim. Acta, Part A 2005, 62, 1203-1208. (51) Demchenko, A. P. Luminescence 2002, 17, 19-42.
Figure 5. Far-UV circular dichroism spectra of BSA at pH 3.8 (curve a), pH 7.0 (curve c), and pH 9.0 (curve b).
by the changes of Trp emission behavior. Also, the pH of the solution had a distinct effect on this change. On considering the larger contribution of Trp-134 to the fluorescence emission of BSA, we should point out that the changes observed here were more related to the Trp-134 surroundings in the external part of domain I, which is more accessible to the solvent.52 Circular Dichroism (CD) Study. CD spectroscopy is one of the commonly used methods to study protein conformations in solution or adsorbed onto colloidal surfaces. To gain a better understanding in the conformational behavior of BSA in the albumin:gold nanoparticle conjugates, CD spectroscopic measurements were then performed. Figure 5 shows the far-UV CD spectra of native BSA at pH 3.8 (curve a), 7.0 (curve c), and 9.0 (curve b), respectively. The CD spectra of BSA at pH 7.0 exhibited two negative minima in the ultraviolet region at 208 and 222 nm, which is characteristic of an R-helical structure of protein. Since the R-helix is one of the elements of secondary structure, the structure change of albumin then could be evaluated by the content of the R-helical structure (denoted as helicity in the following). The helicity of BSA at pH 3.8, 7.0, and 9.0 was estimated to be 53, 62, and 59%, respectively, according to the method described by Lu et al.,37 and the results were in agreement with that reported previously. The highest helicity occurred at pH 7.0, which corresponded to the normal (N) form of the albumin, with a larger decrease at pH 3.8 (F form) and a smaller decrease at pH 9.0 (B form). This change in the helicity of BSA at different pH values mostly originated from the different conformational states they adopted. Albumin in the N form was known to possess the most compact form, while in both the N-F transition and the N-B transition, the molecule of albumin underwent an expansion.28 Thus, a loss of the secondary structure of the albumin was expected for the F and B forms. Figure 6 shows the CD spectra of native BSA and that in the bioconjugates with increasing the concentration of GNP at pH 3.8 (Figure 6A), 7.0 (Figure 6B), and 9.0 (Figure 6C), respectively. As shown, at either case, the ellipticity values at both 208 and 222 nm were found to decrease in the bioconjugates, which indicated the loss of the R-helical structure of protein after the conjugation with nanoparticles. In addition, with increasing the concentration of GNP in the bioconjugates, these two bands appeared to move together toward the region between 208 and 222 nm. As a result, the CD spectra evolved toward a shape more similar to that typical of β-rich structure, which was possibly an indication of the conformational transition from R-helix to β-sheet structure in the bioconjugates. Previous studies have reported that the protein would lose part of its secondary structure in the bioconjugates at the boundary surface of nanoparticles,10-14 which (52) Ribou, A. C.; Vigo, J.; Viallet, P.; Salmon, J. M. Biophys. Chem. 1999, 81, 178-189.
Changes in Albumin:Gold Nanoparticle Bioconjugates
Langmuir, Vol. 23, No. 5, 2007 2719 Table 2. Results of the Linear Fit to the Plots That Correspond to Figure 7
Figure 6. CD spectra of 2.5 × 10-7 M BSA in the native state (curve a) and in the bioconjugates with different concentrations of GNP in the range of 2 × 10-10 to 1.1 × 10-9 M (curves b-e) at pH 3.8 (A), pH 7.0 (B), and pH 9.0 (C).
Figure 7. Helicity of BSA vs the concentration of GNP in the bioconjugates at pH 3.8 (curve a), pH 7.0 (curve b), and pH 9.0 (curve c).
was also the case in the albumin:gold nanoparticle conjugates. The helicity of albumin in the conjugates versus the concentration of GNP at different pH values was shown in Figure 7. As can be seen, the helicity decreased gradually with increasing the concentration of GNP in the bioconjugates. Since the exact adsorbed amount of albumin in the bioconjugates was unclear at present, it was noteworthy here that the gradual decrease of the helicity with an increase in the GNP concentration could be related to either a stronger structural change at a low degree of surface coverage or a lowering in the signal upon dilution with the nanoparticles. Furthermore, the present results showed that BSA could retain most of its helical structure in the bioconjugates. For example, in the case of pH 7.0 (curve b in Figure 7), in which most biological studies are interested, the helicity that BSA possessed was still 46%, which was nearly 75% of its original structure when the concentration of GNP was up to 1.1 nM. This was particularly important for the further application of the
pH
K (nM-1)
3.8 7.0 9.0
-0.117 -0.135 -0.148
relevant coefficient
standard deviation
0.987 0.984 0.993
0.0106 0.0124 0.0091
albumin:GNP bioconjugates because too much loss of the original structure of the protein in the bioconjugates possibly meant the loss of the biological activity of the protein that, however, was unexpected in most studies especially in the biological applications.53 This retention of original structure of the protein in the bioconjugates was possibly owed to the high curvature of gold nanopaticles. As known, BSA, when binding to the planar gold surface (i.e., in surface plasmon resonance studies where the albumin was often used to bind with a thin gold layer), often undergoes larger changes in the secondary structure.54 However, in the bioconjugates prepared by the metal nanoparticles, the higher curvature of gold nanoparticles favored the retention of the original structure of protein greatly.11,15,55 Thus, most of the protein structure could be retained in the bioconjugates, which then makes the bioconjugated nanomaterials appealing in the future applications. On the other hand, the decrease of the helicity of the albumin was found to be linear with the concentration of GNP in the bioconjugates, and the results of the linear regression at each pH were shown in Table 2. The slope of the obtained line actually could reflect the sensitivity of the conformational change to the concentration of GNP at each pH. As seen in Table 2, the value of K again followed an order of pH 9.0 > pH 7.0 > pH 3.8, which showed that the decrease of the helical structure in the bioconjugates was strongly pH-dependent. Thus, the CD study here further showed that the pH of the medium had a strong influence on the conformational changes of the protein in the bioconjugates. The reason attributed to this pH-dependence possibly lies in the intrinsic conformational state that the albumin adopted at different pH values and the type of association forces (such as electrostatic interactions, hydrophobic interactions, and so on) involved in the formation of the bioconjugates. Fourier Transform Infrared (FTIR) Study. FTIR spectroscopy offers another valuable method to monitor the changes in the secondary structure of proteins.56 The specific stretching and bending vibrations of the peptide backbone in amide I, II, and III bands provide information about different types of secondary structures such as R-helix, β-sheets, turns, and unordered structures (referred to as random coil). Of all the amide bands of the peptide group, amide I, which gives rise to infrared bands in the region between approximately 1600 and 1700 cm-1,has been proven to be the most sensitive probe of protein secondary structure.57 A critical step in the IR study of proteins is the assignment of the amide I component bands to different types of secondary structure, and a rough assignment that is suggested in most protein studies is as follows: 1651-1658 cm-1 (R-helix); 1618-1642 cm-1 (β-sheets); 1666-1688 cm-1 (turns); and 1650 ( 1 cm-1 (random coil).13,17,38 (53) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233-244. (54) Silin, V.; Weetall, H.; Vanderah, D. J. J. Colloid Interface Sci. 1997, 185, 94-103. (55) Vertegel, A. A.; Sigel, R. W.; Dordick, J. S. Langmuir 2004, 20, 68006807. (56) Mantsch, H. H.; Chapman, D. Infrared Spectroscopy of Biomolecules; Wiley: New York, 1996. (57) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry 1993, 32, 389-394.
2720 Langmuir, Vol. 23, No. 5, 2007
Figure 8. Original infrared spectra (A) and corresponding second derivative spectra (B) in the amide I region of BSA and BSA:GNP bioconjugates at pH 7.0.
Figure 8 A shows the original FTIR spectra of native BSA and that in the bioconjugates (denoted as BSA:GNP in the figures) at pH 7.0, where an intense band in the amide I region could be observed. As shown, as compared with that for the native protein, this band in the bioconjugates showed obvious changes in both shape and peak position, which suggested the occurrence of changes in the secondary structure of the albumin in the conjugates. More detailed information about this conformational change was then obtained by analyzing the second derivative spectrum of Figure 8A as shown in Figure 8B. A predominant band centered at 1655 cm-1 relative to the other bands in the amide I region indicated a higher content of R-helix structures of BSA at pH 7.0. As shown, the intensities of the bands assigned to β-sheet (1624, 1633, and 1639 cm-1) and turn (1672 cm and 1682 cm-1) structures17,38 for the native BSA were found to increase in the bioconjugates, while that assigned to R-helix structure (1655 cm-1) decreased relatively. The appearance of a band at 1648 cm-1 in the bioconjugates indicated the increase of unordered structures.58 Moreover, almost all the bands shifted to higher frequencies for the conjugates as shown in Figure 8 B. These changes in the IR spectra of albumin after the conjugation with nanoparticles showed that the secondary structure of BSA underwent obvious changes in the BSA:GNP conjugates at pH 7.0. The second derivative spectra further indicated the increase of the sheets, turn, and unordered structures, whereas the content of helical structures decreased. The conformational changes here implied that on the surface of nanoparticles, the albumin would adopt a more incompact conformation state. FTIR studies at pH 3.8 (Figure 9) and 9.0 (Figure 10) were then conducted to investigate the pH effect on the conformational changes. Several similar changes could be observed from the second derivative spectra. First, the intensities of the bands that were assigned to β-sheets and turns increased. As can be seen, in some cases, due to the substantial increase of the bands, several adjacent bands were found to be combined into one band. For example, in Figure 9B, the bands at 1633 and 1641 cm-1 for the native BSA evolved into an intense band centered at 1637 cm-1 in the bioconjugates. Then, the intensity of the vibrational band that was assigned to the R-helical structures of the albumin decreased in both cases when conjugated with nanoparticles, which was in agreement with the case at pH 7.0. Meanwhile, substantial differences with regard to the changes in the IR spectra of the bioconjugates still existed among different pH values. For example, at pH 3.8 (seen in Figure 9B), the band at 1649 cm-1 disappeared in the BSA:GNP conjugates, which indicated a loss (58) Murayama, K.; Tomida, M. Biochemistry 2004, 43, 11526-11532.
Shang et al.
Figure 9. Original infrared spectra (A) and corresponding second derivative spectra (B) in the amide I region of BSA and BSA:GNP bioconjugates at pH 3.8.
Figure 10. Original infrared spectra (A) and corresponding second derivative spectra (B) in the amide I region of BSA and BSA:GNP bioconjugates at pH 9.0.
of unordered structures at pH 3.8, while at pH 7.0 (Figure 8B), an increase of this structure component was found, and at pH 9.0 (Figure 10B) no band assigned to the unordered structures was observed. Moreover, the intensity of the band attributed to the helical structures relative to that of other components and the extent of the increase on the sheet structures in the bioconjugates both showed obvious pH-dependence. The previous results from the IR study clearly suggested that the pH of the medium did influence the conformational changes of albumin in the bioconjugates, which further supported the results of the fluorescence and CD studies.
Conclusion In conclusion, in this work, we studied protein conformational changes in the BSA:gold nanoparticle conjugates by UV-vis, fluorescence, CD, and FT-IR spectroscopic techniques. The studies presented here demonstrated that BSA in the bioconjugates underwent identifiable conformational changes on both the secondary and the tertiary structure levels. Fluorescence studies showed that Trp residues in the albumin were placed in a more hydrophobic environment and confronted more restrictions from their surroundings in the bioconjugates. CD studies suggested a decrease of the helical structures and a possible increase of β-sheet structures in the albumin secondary structure after the conjugation. Also, the decrease of ellipticity at 208 nm was found to be linear with the concentration of gold nanoparticles in the bioconjugates. FTIR studies further indicated the substantial increase of sheet and turn structures accompanied by the decrease of helical structures of BSA in the bioconjugates. Particularly,
Changes in Albumin:Gold Nanoparticle Bioconjugates
the pH effect on the conformational changes of BSA in the bioconjugates was investigated, and the results showed that the pH of the medium influenced the conformational changes greatly. While the IR study showed that the detailed changes of the protein conformation varied at different pH values, both fluorescence and CD studies suggested that the extent of change was larger at higher pH values in the experimental range. This pH-
Langmuir, Vol. 23, No. 5, 2007 2721
dependence possibly originated from the intrinsic conformational state that the albumin adopted at different pH values. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20427003 and 20575064). LA062064E
