Effect of Colloidal Gold Size on the Conformational Changes of

Biomacromolecules 2005, 6, 46-53

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Effect of Colloidal Gold Size on the Conformational Changes of Adsorbed Cytochrome c: Probing by Circular Dichroism, UV-Visible, and Infrared Spectroscopy Xiue Jiang, Junguang Jiang, Yongdong Jin, Erkang Wang, and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022 China Received April 28, 2004; Revised Manuscript Received September 9, 2004

The conformational changes of bovine heart cytochrome c (cyt c) induced by the adsorption on gold nanoparticles with different sizes have been investigated by electronic absorption, circular dichroism (CD), and Fourier transform infrared spectra. The combination of these techniques can give complementary information about adsorption-induced conformational changes. The results show that there are different conformational changes for cyt c adsorbed on gold nanoparticles with different sizes due to the different interaction forces between cyt c and gold nanoparticles. The colloidal gold concentration-dependent conformation distribution curves of cyt c obtained by analysis of CD spectra using the singular value decomposition least-squares method show that the coverage of cyt c on the gold nanoparticles surface also affects the conformational changes of the adsorbed cyt c. Introduction The integration of nanotechnology with biology creates a new subject: nanobiology, which produces many advances in molecular diagnostics, modern materials science, and nanobiosensors.1-5 Recent researches are mainly concerned with bioconjugated nanoparticles for generating novel materials2,6-10 and detection of biomolecules.11-14 Two main methods have been used for bioconjugation: one is using the functional nanoparticles to covalently link biological molecules6,15-17 and the other is by electrostatic interactions.18-20 Both of the methods are connected with the adsorption of protein on a solid surface. The success of these applications depends on the activity of the adsorbed protein being similar to that in its native state, whereas the key feature that determines the activity of the adsorbed protein is the conformation. When protein is adsorbed on a solid surface, its conformation will be changed due to the electrostatic and/or hydrophobic interactions between protein and solid surface, whose physicochemical properties affect strongly the structure rearrangements of the adsorbed protein.21,22 Although many interesting studies have been focused on this subject, there are few reports about how the conformation of the biomolecule is affected by a solid boundary surface of nanoparticles.22-26 However, a detailed understanding of the protein adsorption mechanism will open a wide opportunity for studying the basic physics and chemistry of bioconjugated nanoparticles, and also, it will provide the essential information for the optimization of conditions in all such cases. Cyt c has many advantages as a model for studying conformational changes when it is adsorbed on the surface of nanoparticles. Since its special biological functions (to * To whom correspondence should be addressed. Fax: 86-431-5689711. E-mail: [email protected].

act as an electron carrier in the respiratory chain of an aerobic organism and play an important role in programmed cell death27,28) relate to the conformational changes, several conformational states have been characterized during extensive studies for its binding to anionic surfaces of phospholipids vesicles, micelles, polyanions, and electrodes.27-33 Furthermore, the effects of the interior groups, such as heme and thioether bond, on the conformation of cyt c have also been studied.34,35 Thus, these states can be used as reference states in comparison with the conformation of the adsorbed cyt c on gold nanoparticles with different sizes. In this paper, the conformational changes of cyt c that resulted from the adsorption on colloidal gold nanoparticles with different sizes have been studied by CD, FTIR, and UVvis techniques. Interestingly, we found that conformational changes of cyt c were dependent on the size of colloidal gold nanoparticles and the coverage of adsorbed cyt c on nanoparticles. Furthermore, it is also intended to probe the adsorption mechanism of the protein on nanoparticles surface. Experimental Section Materials. Bovine heart cyt c, purchased from Sigma, was prepared in a 0.1 M phosphate buffer solution (PBS) (pH 7.2). The concentration of the cyt c solution was measured spectrophotometrically using a molar absorptivity of 106 100 mol-1 cm-1 at 410 nm.36 PBS was prepared by Na2HPO4‚ 12H2O and NaH2PO4‚2H2O, doubly purified water was from Milli-Q system. Chloroauric acid trihydrate (HAuCl4‚3H2O) was purchased from EM Sciences. All of the other chemicals were of analytical grade and were used as received. Preparation of Colloidal Gold. All glassware used was cleaned in a bath of freshly prepared 3:1 HCl:HNO3 and rinsed thoroughly in H2O prior to use. Colloidal gold solutions of ca. 16 and 41 nm in diameter were prepared by

10.1021/bm049744l CCC: $30.25 © 2005 American Chemical Society Published on Web 11/06/2004

Conformational Changes of Adsorbed cyt c

citrate-reduction of HAuCl4 as previously described.37 Preparation of “seed colloid” gold solution of ca. 2-4 nm-diameter particles was performed by adding 1 mL of 1% aqueous HAuCl4‚3H2O to 100 mL of H2O with vigorous stirring, followed by the addition of 1 mL of 1% aqueous sodium citrate 1 min later. After an additional 1 min, 1 mL of 0.075% NaBH4 in 1% sodium citrate was added. The solution was stirred for 5 min and then stored at 4 °C.38,39 Samples for Spectrophotometric Determination. The samples for spectroscopic determination were mixtures of cyt c nanoparticles. They were made by diluting cyt c and colloidal gold stock solutions with pH 7.2 PBS under gentle stirring at 4 °C and then stored at 4 °C before measurement. Instruments and Methods. A Cary 500 UV-vis-NIR spectrometer (Varian Co., USA) was used for UV-visible spectra measurements. CD spectra were measured with an AVIV 62A DS circular dichroism spectrometer (AVIV Co., USA) equipped with a thermoelectric temperature control unit, and the spectra were recorded at 25 °C. FTIR spectra were recorded by a Nicolet 520 FT-IR spectrometer (Nicolet Co., USA). The sample solutions were injected in an IR cell for aqueous with CaF2 windows and a 6-µm spacer. For each spectrum, a 300-scan interferogram was collected in a singlebeam mode with a 4-cm-1 resolution. The sample spectra were obtained according to previously established criteria and the double-subtraction procedure.40,41 Second-derivative spectra were obtained with the derivative function of Omnic E. S. P. software. A protein-free nanoparticles suspension was used to record the baseline for mixtures of cyt c nanoparticles in our experiments unless indicated otherwise. Activity and Stability Assay of cyt c. The activity and stability of the cyt c adsorbed at both 16- and 2-4-nm nanoparticles were determined by in situ UV-visible spectroelectrochemical experiments and UV-visible spectra measurements. The spectroelectrochemical experiments were carried out in a homemade long optical path thin layer cell (LOPTLC), Cary 500 UV-vis-NIR spectrometer (Varian Co., USA) for UV-visible spectrum measurement, and a CHI 630 electrochemical instrument (CHI Co. USA) for electrochemical operation. A piece of glassy carbon (8 mm × 8 mm) was used as the working electrode for in situ spectroelectrochemical experiments, a platinum wire as the auxiliary electrode, and Ag/AgCl (saturated KCl) as the reference electrode. Before the experiment, the working electrode was mechanically polished with 1.0-, 0.3-, and 0.05-µm R-Al2O3 slurry, successively, and washed ultrasonically in doubly purified water followed by ethanol for a few minutes for each step, and then the freshly polished electrode was scanned over the potential range of 0.0-1.5 V (vs Ag/ AgCl) for 50 segments at a scan rate 0.1 V/s. So the electrode surface was functioned by the negative charge derived from carbon derivatives, such as -COO-. The LOPTLC was constructed with a 10.0-mm optical path length and 0.2-mm thickness of thin layer. Data Analysis (Singular Value Decomposition Least Squares Analysis (SVDLS)). To ascertain the different conformations of cyt c presented over a range of colloidal gold concentrations, the CD spectral data were evaluated by

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Figure 1. Solide lines: the electronic absorption spectra of mixtures between cyt c and different concentrations of 16-nm gold sol. 3.5 µM cyt c was mixed with (a) 0, (b) 1.6, (c) 3.3, (d) 4.9, (e) 6.5, (f) 8.2, (g) 9.8, (h) 11.5, (i)12.8, (j) 14.7, and (k) 18 nM gold sol in 0.1 M PBS (pH 7.2). Dotted line: absorption spectra of gold sol stock solution. Dashed line: absorption spectra of 18 nM gold sol in 0.1 M PBS (pH 7.2).

SVDLS analysis. In this analysis, several CD spectra were measured with the same m (m ) total wavelengths) wavelengths. The spectra were grouped into series taken under identical conditions except for the changes of a single variable (as the colloidal concentration). The SVDLS mathematical and computational details have been reported previously.42,43 Results Adsorption of cyt c on Colloidal Gold Nanoparticles. The lowest saturated monolayer adsorption concentration of cyt c on colloidal gold was determined spectrophotometrically by finding the optimal concentration of colloidal gold that cyt c just could not prevent electrolyte-induced aggregation of the gold sol. It is known that the surface plasmon resonance of isolated gold particles is at about 520 nm (Figure 1, dotted line); however, when gold sol is added into 0.1 M PBS (pH 7.2), a broad absorbance due to the aggregation of the gold sol at long wavelength (∼ 677 nm) (Figure 1, dashed line) can be observed,44 whereas the absorbance at 520 nm becomes broad accompanied by red shift to 537 nm. This is because the electrolytes can cause particle flocculation due to screening of the repulsive double layer charges that normally stabilize them. However, this flocculation can be prevented if sufficient cyt c exists in PBS; the adsorbed cyt c can stabilize the isolated gold sol due to its steric repulsions between the particles.19 So we can probe the lowest concentration ratio of cyt c to nanoparticles according to the appearance of the aggregation peak of nanoparticles. Figure 1 (solid lines) shows the electronic absorbance spectra of the mixtures of cyt c (3.5 µM) with various concentrations of gold sol in 0.1 M PBS (pH 7.20) (all of the spectra were recorded based on PBS which was used to record the blank). Compared with the absorbance of cyt c (Figure 1, curve a), the Q-band shows an apparent broadening and red shift with addition of gold sol for the interaction of cyt c with gold sol, and at the same time, a broad and very

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Figure 2. (A) Absorption spectra of mixtures between cyt c and different concentrations of 16-nm gold sol. 3.5 µM cyt c was mixed with (1) 0, (2) 1.6, (3) 3.3, (4) 4.9, (5) 6.5, (6) 8.2, (7) 9.8, (8) 11.5, (9) 14.7, and (10) 18 nM gold sol in 0.1 M PBS (pH 7.2). (B) The absorption spectra of mixtures between cyt c and different concentrations of 2-4-nm gold sol. 3.5 µM cyt c was mixed with (1) 0, (2) 11.2, (3) 22.4, (4) 33.6, (5) 44.8, (6) 78.4, (7) 100.8, (8) 123.2, (9) 145.6, and (10) 168 nM gold sol in 0.1 M PBS (pH 7.2).

weak absorbance at about 630 nm for the aggregation of gold nanoparticles can be observed when the addition of gold sol was increased to 18 nM (Figure 1, curve k). This indicates that all of the cyt c in the solution has been adsorbed completely on colloidal gold nanoparticles. Thus, the lowest ratio of cyt c to 16 nm gold particles that prevents colloidal gold aggregation was about 200 cyt c per gold nanoparticle. Similarly, the lowest ratio of cyt c to 2-4-nm gold particles can also be estimated as about 20 cyt c per gold nanoparticle. Figure 2 shows the adsorption-induced electronic absorption spectra of cyt c after addition of different concentrations of 16-nm (A) and 2-4-nm (B) gold particles. It can be seen that, with the increase in the concentration of colloidal gold,

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Figure 3. (A) CD spectra of mixtures between cyt c and different concentrations of 16-nm gold sol. 3.5 µM cyt c was mixed with (a) 0, (b) 1.6, (c) 3.3, (d) 4.9, (e) 6.5, (f) 8.2, (g) 9.8, (h) 11.5, (i) 14.7, and (j) 18 nM gold sol in 0.1 M PBS (pH 7.2). (B) CD spectra of cyt c obtained after treating the CD spectra shown in Figure 3A by SVDLS analysis: (a-e): parallel β-sheet, random coil, antiparallel β-sheet, R-helix, and β-turn. (C) The 16-nm colloidal gold concentrationdependent conformational distribution curves of cyt c obtained by SVDLS analysis, (a-e): parallel β-sheet, random coil, antiparallel β-sheet, R-helix, and β-turn.

the absorption intensity of cyt c in Soret band at 410 nm decreases regardless of the effect of colloidal gold size. However, the Q-band shows a red-shift of 4-534 nm with a decrease in absorption intensity and an appearance of a shoulder peak at 548 nm when cyt c is adsorbed on 16-nm gold particles (Figure 2A, insert), whereas a blue shift from 530 to 522 nm with an increase in intensity is observed when cyt c is adsorbed on 2-4-nm gold particles (Figure 2B, insert). The different changes of the Q-band resulted from the different sizes of gold nanoparticles that reflect the different changes of heme microenvironment for the adsorbed cyt c. On the other hand, the colloidal gold concentrationdependent changes of absorption spectra indicate that the coverage of cyt c on the gold surface will also affect the conformation of the adsorbed cyt c. We also have investigated the absorption spectra of cyt c after addition of 41-nm gold particles and obtained similar results as that of cyt c adsorbed on 16-nm gold particles. So



Figure 5. (A) Soret-CD spectra of 3.5 µM cyt c in 0.1 M PBS (pH 7.2) after addition of (1) 0, (2) 18 nM, 16-nm colloidal gold. (B) The Soret-CD spectra of 3.5 µM cyt c in 0.1 M PBS (pH 7.2) after addition of (1) 0, (2) 168 nM, 2-4-nm colloidal gold.

Figure 4. (A) CD spectra of mixtures between cyt c and different concentrations of 2-4-nm gold sol. 3.5 µM cyt c was mixed with (a) 0, (b) 11.2, (c) 22.4, (d) 33.6, (e) 44.8, (f) 56, (g) 78.4, (h) 100.8, (i) 123.2, (j) 145.6, and (k) 168 nM gold sol in 0.1 M PBS (pH 7.2). (B) CD spectra of cyt c obtained after treating the CD spectra shown in Figure 4A by SVDLS analysis: (a-e): R-helix, random coil, β-turn, antiparallel β-sheet, and parallel β-sheet. (C) The 2-4-nm colloidal gold concentration-dependent conformational distribution curves of cyt c obtained by SVDLS analysis, (a)∼(e): R-helix, random coil, β-turn, antiparallel β-sheet, and parallel β-sheet.

we focus on the size effect study by using 2-4-nm and 16nm gold particles on the adsorbed cyt c in the following experiments. CD Spectra Study. Figure 3A shows the far-UV CD spectra of cyt c after addition of 16 nm colloidal gold. It can be seen that the negative Cotton peak decreases gradually with the concentration of 16-nm colloidal gold increasing from 0 to 18 nM. Figure 3B shows the spectra of five different conformations of cyt c45 obtained after treating the CD spectra shown in Figure 3A by SVDLS analysis. According to the Gauss-Markoff mode of protein secondary structure CD spectra46 and the reported results,40 curves a, b, c, d, and e are attributed to parallel β-sheet, random coil, antiparallel β-sheet, R-helix, and β-turn, respectively. Figure 3C exhibits the corresponding colloidal gold concentration-

dependent fractional distribution plot for the five conformations shown in Figure 3B. As can be seen, the different secondary structures of native cyt c, i.e., parallel β-sheet, random coil, R-helix, and β-turn, coexist in solution with fractions of 0.11, 0.30, 0.35, and 0.24, respectively. With the addition of colloidal gold, the fractions of random coil (curve b), R-helix (curve d), and β-turn (curve e) decrease gradually to 0.06, 0.007, and 0.11, respectively, and the antiparallel β-sheet (curve c) fraction increases to 0.36, whereas for parallel β-sheet (curve a), its fraction first decreases and then increases to 0.46 until cyt c is adsorbed completely on gold nanoparticles. This implies that the conformational transitions of cyt c are mainly from R-helix, random coil, and β-turn to β-sheet, which results in a more incompact conformation. Also, Figure 3C indicates clearly that the coverage of cyt c on 16-nm gold surface can affect the conformation of the adsorbed cyt c. Figure 4A shows the far-UV CD spectra of cyt c after addition of 2-4-nm colloidal gold. Unlike CD spectra changes of cyt c induced by 16-nm colloidal gold, the negative Cotton peak increases slightly with the addition of 2-4-nm colloidal gold. This implies that conformational changes of cyt c are influenced heavily by the size of gold nanoparticles. The CD spectra of the five different conformations are extracted from the CD spectra shown in Figure 4A by SVDLS analysis (Figure 4B). They are attributed to R-helix (curve a), random coil (curve b), β-turn (curve c), antiparallel β-sheet (curve d), and parallel β-sheet (curve e),46 respectively. The corresponding colloidal gold concentrationdependent fractional distribution plot for the five conforma-

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Figure 6. Infrared spectra of cyt c (4 mM) in 0.1 M PBS, pH 7.2, in the absence (solid line) and presence (dotted line) of 16-nm (A) and 2∼4-nm (B) colloidal gold nanoparticles (10 nM) and corresponding second derivative amide I spectra (A′) and (B′).

tions is shown in Figure 4C. Compared with Figure 3C, different conformational changes are induced when cyt c is adsorbed on 2-4-nm gold particles. Especially in the case of the R-helical fraction, there is a dramatic decrease when native cyt c is adsorbed on 16-nm gold particles, whereas it increases from 0.35 to 0.55 in the presence of 2-4-nm gold particles. This sufficiently proves the effect of colloidal gold size on the secondary structure of the adsorbed cyt c. Comparing with the native conformation of cyt c, a more compact conformation can be induced due to the increase of R-helix and the decrease of random coil when cyt c is adsorbed on 2-4-nm gold particles. Furthermore, just like that of the cyt c adsorbed on 16-nm gold particles, the coverage-dependent conformational changes of cyt c adsorbed on 2-4-nm gold particles can also be seen from Figure 4C. The Soret-CD spectra of native cyt c in PBS display an S-shaped feature with a minimum and maximum at about 418 and 402 nm (Figure 5A, curve 1, and Figure 5B, curve 1), respectively. The addition of 16-nm colloidal gold results in the decrease of negative and positive bands in intensity accompanied by a 3-nm red-shifting of positive peak for native cyt c (Figure 5A, curve 2). However, only the decrease in intensity of negative and positive peaks is induced due to the addition of 2-4-nm gold particles (Figure 5B, curve 2). These differences indicate that gold nanoparticles with different sizes show different effects on heme microenvironment.

FTIR Spectra Study. It is know that, among nine characteristic vibrational bands or group frequencies that arise from the amide groups of protein, the amide I vibrational band, which is almost entirely due to the CdO stretch vibrations of the peptide linkages, is the most sensitive probe of protein secondary structure. The original difference spectra of cyt c in 0.1 M pH 7.2 PBS in the absence and presence of 16- and 2-4-nm gold nanoparticles are shown in Figure 6, parts A and B, respectively. The spectra contain bands for amide I (1700-1600 cm-1) and amide II (1600-1500 cm -1), and more detailed information about the secondary structure of protein can be obtained from the secondderivative analysis of amide I.40,41,47 The amide I band frequency assignments for secondary structures of cyt c in water solution are as follows: R-helix (1656(2 cm-1), unordered (1650(1 cm-1), β-sheets (multiple bands between 1642 and 1624 cm-1), and turns (multiple bands between 1688 and 1666 cm-1).41 Figure 6A′ shows the second derivative spectra of cyt c in 0.1 M PBS in the absence (solid line) and presence (dotted line) of 16-nm gold particles. As can be seen, there are two major bands at 1639 cm-1 (β-sheet) and 1672 cm-1 (β-turn) (Figure 6A′, solid line) for cyt c, whereas with the addition of 16-nm gold particles, these bands disappear, indicating the decrease of β-turn and β-sheet structures. To investigate the effect of colloidal size on the conformational changes



cyt c adsorbed on gold sol with different sizes, we focused on the changes of Soret band during the redox process because the interaction of cyt c with gold sol will affect the Q-band as discussed above. As shown in Figure 7, the absorbance intensity increases about 11%, 8.8%, and 7.3% with a red shift of 5, 4, and 3.5 nm in the Soret band for cyt c (A) and cyt c adsorbed on on 2-4-nm (B) and 16-nm (C) gold sol upon redox process, respectively. So the activity of cyt c adsorbed on 2-4-nm gold sol is higher than that adsorbed on 16-nm gold sol. We also determine the stability of cyt c adsorbed on 16- and 2-4-nm gold nanoparticles according to the change of absorbance intensity of adsorbed cyt c in Soret band versus time. An apparent decrease in absorbance intensity of Au-cyt c could be observed when the protein is incubated with nanoparticles for about 1 h, after that, the absorbance intensity in the Soret band was almost unchanged, indicating that the complexes of Au-cyt c become stable. Discussion

Figure 7. In situ UV-vis spectra of cyt c (A) and adsorbed cyt c on 2-4-nm (B) and 16-nm (C) gold sol at GCE in 0.1 M PBS (pH)7.2), solid line: at 0.6 V, dotted line, at -0.3 V.

of adsorbed cyt c, the second derivative spectra of cyt c in the absence (solid line) and presence (dotted line) of 2-4nm gold particles are shown in Figure 6B′. It can be seen that two main absorption bands at 1681 (β-turn) and 1633 cm-1 (β-sheet) increase obviously after addition of 2-4-nm gold nanoparticles (Figure 6B′, dotted line) compared with that of cyt c (Figure 6B′, solid line), indicating the increase of β-turn and β-sheet structures. On the other hand, addition of 16-nm gold particles can induce a 5-cm-1 blue shift of the characteristic group frequencies (Figure 6A′, dotted line), whereas a 1-cm-1 red shift was induced by 2-4-nm colloidal gold (Figure 6B′, dotted line). This indicates that the size effect of nanoparticles is apparent on intramolecular hydrogen bond of the adsorbed cyt c. Thus, there is an opposite effect of gold nanoparticles with different sizes on the conformational changes of the adsorbed cyt c. Activity and Stability of the Adsorbed cyt c. Figure 7 shows in situ UV-vis spectra of cyt c (A) and adsorbed cyt c on 2-4-nm (B) and 16-nm (C) gold sol at GCE in 0.1 M PBS (pH)7.2), respectively. To investigate the activity of

The changes of a protein three-dimensional structure will be induced when it is adsorbed onto a nanoparticle surface. However, the size effect on the interaction between the protein and solid surface is still not identified clearly despite the wide interest and practical importance of this phenomenon. In this paper, we combined UV-vis, CD, and FTIR spectroscopic techniques to study the above-mentioned phenomenon taking the adsorption of cyt c on the surface of gold nanoparticles as an example to get some information at the molecular level. The intense Soret band and Q-band result from the π-π* transitions of the porphyrin ring in cyt c. The additive effects of the transition dipole moments between two orbital excitations, a2u -b1u and a2u-eg, will affect the intense Soret band and Q-band, respectively.48 Therefore, the intensity of the Soret and Q-bands will be affected by changes in the symmetry of the porphyrin ring.49 The similar changes for the Soret band upon the addition of 16- or 2-4-nm gold particles may reflect the similar heme microenvironment changes of adsorbed cyt c (Figure 2). However, this conclusion is not supported by the changes of the Q-band. The red-shifting of the Q-band accompanied with the decreasing of the absorption intensity and the appearance of a shoulder peak upon the addition of 16-nm gold particles (Figure 2A) are similar to the results reported by Rosell et al.35 The authors had compared the spectrum of Cys14Ser with that of native cyt c. They found that the Soret bands of the two proteins were nearly superimposable and the Q-band of Cys14Ser shifted about 4 nm to a lower energy accompanied with the decreasing of intensity and appearance of a shoulder peak. Taking into account this result, it is reasonable to propose that the thioether bonds formed by Cys14 and Cys17 with a heme prosthetic group are affected heavily due to the adsorption of cyt c on the surface of 16nm gold particles. Therefore we can deduce that the electrostatic reaction sites of cyt c are located at the right groove. The right groove is formed by two R-helixs and peptide 12∼20, which is a positively charged surface enriched by lysine. On the contrary, the blue shifting of the Q-band accompanied with the increasing in intensity can be

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observed by addition of 2-4-nm gold particles. This may imply the formation of high-spin (HS) species because these phenomena are similar to those induced by addition of GuHCl into native cyt c solution.27 However, this conclusion is not consistent with the changes of the Soret CD spectrum (Figure 5B) because the formation of the HS species corresponds to a single positive band at about 408 nm with relatively intensity.27 So this phenomenon may result from the increasing of cyt c hydrophobic nature due to the increase of the R-helix and the decrease of the random coil when cyt c is adsorbed on 2-4-nm gold particles (Figure 4C). The hydrophobic interaction is also an important interaction force between the protein and solid surface accompanied with the conformational changes.26 We can conclude that the hydrophobic interactions might be the main force inducing the adsorption of cyt c on 2-4-nm gold particles. The Soret CD spectrum originating from the coupling of the electronic transition dipole moments of the heme and nearby aromatic amino acids27 is considered to represent a fingerprint for the integrity of the heme pocket. So the changes in the Soret CD spectrum should specifically reflect microenvironment perturbations of this part of the heme pocket that cannot be observed by UV-vis spectra. The negative Cotton effect at 418 nm has been ascribed to the Phe82 and Met-80-heme interaction, and the positive Cotton peak reflects the states of axial heme bonds.50 The difference of changes in Soret CD spectra suggests the different microenvironment disturbances on the upper part of the heme pocket of the adsorbed cyt c due to the different interaction forces between cyt c and gold nanoparticles with different sizes. Therefore, major tertiary structure changes should be different, which probably also give rise to the different changes of far-UV CD spectra (Figures 3 and 4). This also can be proved by the opposite changes of FITR spectra (Figure 6). The fractional changes of five conformations upon the addition of colloidal gold (Figures 3C and 4C) indicate that the different coverages of cyt c on the gold surface will affect the conformation of the adsorbed cyt c. Zhou et al.23 have introduced the concept of effective coverage to describe the status of adsorbed cytochrome b-562 on the surface of gold particles taking into account factors such as the shape of the protein and the protein electrostatic repulsive force. They found that the “side-on” conformation predominated at low cytochrome b-562 concentration, whereas the “tail-on” conformation predominated at high concentration. Considering the results reported by Zhou and co-workers23 and our experimental results as described in the section of CD spectra study, it is reasonable to think that the “side-on” conformation is an incompact conformation when cyt c is adsorbed on 16-nm gold, whereas it is a compact conformation when cyt c is adsorbed on 2-4-nm gold. According to the lowest ratio of cyt c to nanoparticles as discussed above and the surface area of per nanoparticle, we can calculate approximately the protein per surface area of the compact and the incompact conformation adsorbed on 2-4- and 16-nm gold sol are about 2.5 and 4.2 nm2, respectively.

Jiang et al.

Conclusions In conclusion, the effect of colloidal gold size on the conformational changes of the adsorbed cyt c was studied by CD, UV-vis, and FTIR spectroscopic techniques. The main points of this study are summarized as follows: 1. A more compact conformation was induced when cyt c was adsorbed on 2-4-nm gold particles than that adsorbed on 16-nm gold particles, and the activity of cyt c adsorbed on 2-4-nm gold particles is higher than that adsorbed on 16-nm gold particles. 2. The electrostatic interactions were probably the main force causing the adsorption of cyt c on 16-nm gold particles, whereas the hydrophobic interactions were probably the main force for the adsorption of cyt c on 2-4-nm gold particles compared with electrostatic attraction. 3. The thioether bonds formed by Cys14 and Cys17 with the heme prosthetic group were affected heavily by the adsorption of cyt c on the surface of 16-nm gold particles, and one of the adsorption sites for cyt c was right groove. 4. The adsorption of cyt c on nanoparticles with different sizes had different effects on intramolecular hydrogen bonding. 5. The conformational changes of the adsorbed cyt c were related to the coverage of cyt c on nanoparticles. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Nos. 20275037, 20275036, and 20211130506). References and Notes (1) Lavan, D. A.; Lynn, D. M.; Langer, R. Nat. ReV. Drug DiscoVery 2002, 1, 77-84. (2) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4129-4158. (3) Mckenzie, K. J.; Marken, F. Langmuir 2003, 19, 4327-4331. (4) Brown, K. R.; Fox, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1154-1157. (5) Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504-509. (6) Reynolds, R. A., III.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-3796. (7) Storhoff, J. J.; Mirkin, C. A. Chem. ReV. 1999, 99, 1849-1862. (8) Boal, A. K.; Ilhan, F.; Derouchey, J. E.; Thurn-Albrecht, T.; Russell, T. P.; Rotello, V. M. Nature 2000, 404, 746-748. (9) Li, M.; Schnablegger, H.; Mann, S. Nature 1999, 402, 393-395. (10) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (11) Maxwell, D. J.; Taylor, J. R.; Nie, S. J. Am. Chem. Soc. 2002, 124, 9606-9612. (12) Alivisatos, A. P. J. Phys. Chem. 1996, 100, 13226-13239. (13) Bawendi, M. G. Solid State Commun. 1998, 107, 709-711. (14) Parak, W. J.; Pellegrino, T.; Micheel, C. M.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. Nano Lett. 2003, 3, 33-36. (15) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665-668. (16) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (17) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142-12150. (18) Shenton, W.; Davis, S. A.; Mann, S. AdV. Mater. 1999, 11, 449452. (19) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phy. Chem. B 1998, 102, 9404-9413. (20) Mahtab, R.; Rogers, J. P.; Murphy, C. J. J. Am. Chem. Soc. 1995, 117, 9099-9100. (21) Karlsson, M.; Mårtensson, L. G.; Jonsson, B. H.; Carlsson, U. Langmuir 2000, 16, 8470-8479. (22) Norde, W. AdV. Colloid Interface Sci. 1986, 25, 267-340. (23) Zhou, H. S.; Aoki, S.; Honma, I.; Hirasawa, M.; Nagamune, T.; Komiyama, H. Chem. Commun. 1997, 605-606.

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Effect of Colloidal Gold Size on the Conformational Changes of

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