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Interaction of Apo Cytochrome c with Sulfonated Polystyrene Nanoparticles Li Liang, Ping Yao,* Jie Gong, and Ming Jiang Department of Macromolecular Science and the Key Laboratory of Molecular Engineering of Polymer, Fudan University, Shanghai 200433, China Received October 3, 2003. In Final Form: January 26, 2004 Stable nanoparticle dispersion in aqueous solutions was obtained with partially sulfonated polystyrene. The hydrophobic association of the backbone chains and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The size distribution of the sulfonated polystyrene particles in relation to concentration, degree of sulfonation and chain length, and pH was characterized by dynamic laser light-scattering. The structure and morphology of the particles were characterized with fluorescence and atom force microscopy. Highly sulfonated polystyrene particles can form large complex particles with positively charged protein, apo cytochrome c. Dynamic laser light-scattering and atom force microscopy studies show that the size and distribution of the complex particles depend on the relative amount of apo cytochrome c and sulfonated polystyrene. When sulfonated polystyrene is in excess, apo cytochrome c interacts with sulfonated polystyrene particles forming stable complexes and excessive sulfonated polystyrene particles bind to the periphery of the complexes preventing them from further aggregation. When apo cytochrome c is in excess, apo cytochrome c links the complexes forming much larger particles. Fluorescence study demonstrates that the hydrophobicity/hydrophility of the complex particles is relative to the ratio of apo cytochrome c and sulfonated polystyrene, degree of sulfonation, and pH. Apo cytochrome c not only can neutralize the negative charges on the surface of sulfonated polystyrene particles, but may also insert into the cores disrupting the original structure of sulfonated polystyrene particles.
Introduction Lightly sulfonated polystyrene (SPS) has been investigated usually in organic solvents1-4 in the past because they are insoluble in water. In recent years, Jiang and co-workers developed the “microphase inversion” method to produce surfactant-free nanoparticles5-8 composed of the lightly sulfonated polystyrene by mixing dropwise SPS-THF solution with an excess amount of water. Fluorescence and dynamic laser light-scattering studies demonstrated that the ionic groups on the particle surface stabilized the hydrophobic core made of the polystyrene chains. The particle size depended on the degree of sulfonation, the initial concentration of the SPS-THF solution, as well as the mixing order, etc. Essafi et al.9 found that highly sulfonated polystyrene could form hydrophobic domains in water. In this work, we prepared surfactant-free nanoparticles with highly sulfonated polystyrene in aqueous solution. The nanoparticles produced with a different degree of sulfonation (DS) and chain * To whom correspondence should be addressed. Fax: 86-2165640293. E-mail:
[email protected]. (1) Lantman, C. W.; MacKnight, W. J.; Peiffer, D. G.; Sinha, S. K.; Lundberg, R. D. Macromolecules 1987, 20, 1096-1101. (2) Young, A. M.; Higgins, J. S.; Peiffer, D. G.; Rennie, A. R. Polymer 1995, 36, 691-697. (3) Young, A. M.; Garcia, R.; Higgns, J. S.; Timbo, A. M.; Peiffer, D. G. Polymer 1998, 39, 1525-1532. (4) Chakrabarty, K.; Weiss, R. A.; Sehgal, A.; Seery, T. A. Macromolecules 1998, 31, 7390-7397. (5) Li, M.; Jiang, M.; Zhu, L.; Wu, C. Macromolecules 1997, 30, 22012203. (6) Li, M.; Jiang, M.; Wu, C. J. Polym. Sci., Part B: Polym. Phys. 1997, 35, 1593-1599. (7) Zhang, G. Z.; Li, X. L.; Jing, M.; Wu, C. Langmuir 2000, 16, 92059207. (8) Liu, S. Y.; Hu, T. J.; Liang, H. J.; Jiang, M.; Wu, C. Macromolecules 2000, 33, 8640-8643. (9) Essafi, W.; Lafuma, F.; Williams, C. E. J. Phys. II 1995, 5, 12691275.
length of SPS were characterized by steady-state fluorescence, atom force microscopy, and dynamic lightscattering. Cytochrome c (cyt c) plays important roles in electrontransfer chain and programmed cell death. Apo cyt c is the precursor of the mitochondria protein cyt c, which is encoded by nuclear DNA and synthesized on free cytoplasmic ribosomes. Apo cyt c inserts spontaneously and partially crosses the outer mitochondrial membrane. After or simultaneous with translocation across the outer membrane, apo cyt c binds with heme and holo cyt c folds the polypeptide around the heme into the native structure.10,11 In contrast with cyt c, which has a compact welldefined structure in aqueous solution, the heme-free apo cyt c has a disordered structure in solution. The study of the folding kinetic mechanism of apo cyt c in lipid micelles in vitro showed that apo cyt c inserted to the lipid micelle after the formation of helical structure on the surface of the lipid.11 The biophysical basis of how a polypeptide chain selfassembles into a stable, native protein within a biologically relevant time scale has been a problem fascinating theoreticians and experimentalists for decades. The occurrence of diseases that resulted from the structural transformation and misfolding of a peptide has made the study of protein folding one of the most attractive research fields.12-15 The development of a protocol for the efficient refolding of the target recombinant protein has become (10) Rankin, S. E.; Watts, A.; Pinheiro, T. J. T. Biochemistry 1998, 37, 12588-12595. (11) Bryson, E. A.; Rankin, S. E.; Carey, M.; Watts, A.; Pinheiro, T. J. T. Biochemistry 1999, 38, 9758-9767. (12) Gorman, P. M.; Chakrabartty, A. Biopolymers 2001, 60, 381394. (13) DeMager, P. P.; Penke, B.; Walter, R.; Harkany, T.; Hartignny, W. Curr. Med. Chem. 2002, 9, 1763-1780. (14) Zerovnik, E. Eur. J. Biochem. 2002, 269, 3362-3371. (15) Yon, J. M. Braz. J. Med. Biol. Res. 2001, 34, 419-435.
10.1021/la035844l CCC: $27.50 © 2004 American Chemical Society Published on Web 03/06/2004
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an important issue because a variety of genetically engineered proteins for use in the medical and bioindustrial area have been produced as inclusion bodies, which have no biological activity, in the host cells.16 The studies of the interaction of protein and polymer by several research groups have shown that protein folding can be assisted by the polymer assemblies: the microsphere prepared by vinyl polymers modified with the methacryl acid and thiol assisted the refolding of RNaseA;17 poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-PhPEG) nanoassemblies assisted the refolding of bovine carbonic anhydrase;16 and the nanogels of cholesterolbearing pullulan assisted the refolding of carbonic anhydrase and citrate synthase.18 The previous work in our lab found that highly sulfonated polystyrene nanoparticles could interact with apo cyt c, and the interaction led to a conformational transition of apo cyt c from random coil to R-helix.19 Our circular dichroism study found that the R-helix content of apo cyt c induced by SPS particles was dependent on concentration, DS and chain length of SPS, as well as the pH and ionic strength of the solution. However, the mechanism of the folding of apo cyt c induced by sulfonated polystyrene has not been known. In this report, we study the interaction of SPS particles and apo cyt c using a combination of techniques, i.e., dynamic lightscattering, steady-state fluorescence, and atom force microscopy, in order to gain insight into the interaction and its effect on the structure and hydrophobicity, etc., of the particles. Experimental Section SPS Samples. The preparation of highly sulfonated polystyrene samples was described in an earlier report.19 In this paper, two series of SPS were used. (1) xS-PS1 series having low Mw PS of 6100 Da, i.e., average repeating units of 60 and different DS (x) ranging from 19 to 38 mol %. (2) xS-PS2 series with a much higher molecular weight of PS (28 500 Da), i.e., average repeating units of 280 and x from 14 to 55 mol %. Preparation of Stock Solutions. SPS nanoparticle stock solutions with concentrations of 7 × 10-4 g/mL at desired pH values were prepared as reported previously.19 Horse heart cyt c was purchased from Sigma. Apo cyt c was prepared by chemically removing heme group of cyt c as described by Fisher et al.20 The concentration of the stock solution of apo cyt c is 100 µM, which was measured spectrophotometrically with a molar extinction coefficient of 10 580 M-1 cm-1 at 277 nm.21 Samples for Spectrophotometry. SPS stock solution was added dropwise into the solution with the desired pH and NaCl concentration. Then apo cyt c was added dropwise into the SPS solution and stirred at least 12 h for light-scattering and fluorescence measurement. Dynamic Light-Scattering (DLS) Measurements. A commercial laser light-scattering spectrometer (Malvern Autosizer 4700) equipped with a multi-τ digital time correlation (Malvern PCS7132) and Compass 315M-100 Diode-Pumped Laser (output power g 100 mW, CW at λ0 ) 532 nm) as a light source was used. All the DLS measurements were done at 25.0 ( 0.1 °C and at a scattering angle of 90°. The measured time correlation functions were analyzed by Automatic Program equipped with the correlator. (16) Yoshimoto, N.; Hashimoto, T.; Felix, M. M.; Umakoshi, H.; Kuboi, R. Biomacromolecules 2003, 4, 1530-1538. (17) Shimizu, H.; Fujimoto, K.; Kawaguchi, H. Colloid Surf. A 1999, 153, 421-427. (18) Nomura, Y.; Ikeda, M.; Yamaguchi, N.; Aoyama, Y.; Akiyoshi, K. FEBS Lett. 2003, 553, 271-276. (19) Gong, J.; Yao, P.; Duan, H.; Jiang, M.; Gu, S.; Chunyu, L. Biomacromolecules 2003, 4, 1293-1300. (20) Fisher, W. R.; Taniuchi, H.; Anfinsen, C. B. J. Biol. Chem. 1973, 248, 3188-3195. (21) Stellwagen, E.; Rysavy, R.; Babul, G. J. Biol. Chem. 1972, 247, 8074-8077.
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Figure 1. Hydrodynamic diameter (Dh) distributions of 32SPS1 particles in pH 2.0 solutions after a successive dilution. Steady-State Fluorescence Measurements. The fluorescence was measured using a fluorescence spectrophotometer FLS920 (Edinburg Instruments). The spectral resolution of both excitation and emission was 1 nm. Recrystallized pyrene was used as a fluorescence probe, and the final concentration was 2 × 10-7 g/mL. Before the fluorescence measurement, the solutions were stirred for at least 12 h after the addition of pyrene. The emission and excitation spectra were recorded with an excitation and emission wavelength of 335 and 390 nm, respectively. Atom Force Microscopy (AFM) Measurements. The AFM samples were prepared by drying the solution naturally on freshly cleaved mica at room temperature. Image acquisitions were performed in tapping mode on a Nanoscope IV from Digital Instruments equipped with a silicon cantilever with 125 µm and E-type vertical engage piezoelectric scanner.
Results and Discussion Influence of the Concentration on the Dh Distribution of SPS Particles. Figure 1 shows Dh distributions of 32S-PS1 particles in solutions with different concentrations at pH 2.0, which were obtained by successive dilution from a stock solution of 7.0 × 10-4 g/mL. The results show that a concentration changing over two orders of magnitude does not affect the particle size distribution significantly, which is similar to the self-assembly of lightly sulfonated polystyrene,6 carboxy-terminated polystyrene,8 and block copolymers of styrene and sulfonated polyisoprene22,23 in aqueous solution. This implies that the nanoparticles are quite stable against dilution. Influence of pH and Ionic Strength on Dh Distribution of the SPS Particles. A series of 32S-PS1 particle solutions (1.4 × 10-4 g/mL) with different pH values were prepared, and Dh was measured after onehalf of a month storage. Considering the pKa of benzenesulfonic acid, 2.554,24 and the complete dissociation of poly(sodium 4-styrenesulfonate) (PSS), 100% sulfonated polystyrene, in aqueous solution from pH 2.0 to 13.0 (data not shown), we thought that partly sulfonated polystyrene should completely dissociate when pH is higher than 2.0. Therefore, it was expected that the size of SPS particles was independent of pH. However, Figure 2 shows that Dh increases as pH increases. This is similar to the result of polystyrene-block-poly(methacrylic acid) micelles in aqueous media,25 which was explained as the stretching of the chains with negatively charged carboxylic groups as pH (22) Szczubialka, K.; Ishikawa, K.; Morishima, Y. Langmuir 2000, 16, 2083-2092. (23) Szczubialka, K.; Ishikawa, K.; Morishima, Y. Langmuir 1999, 15, 454-462. (24) Dean, J. A. Lange’s Handbook of Chemistry, 15th ed.; Beijing World Publishing Corporation/McGraw-Hill Book Co.: Beijing, 1999; Section 8.31. (25) Karymov, M. A. Langmuir 1996, 12, 4748-4753.
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Figure 2. Hydrodynamic diameter (Dh) distributions of 32SPS1 at different pH solutions. The concentration of 32S-PS1 is 1.4 × 10-4 g/mL.
Figure 3. Hydrodynamic diameter (Dh) of S-PS2 series with different DS at pH 2.0; the concentration is 1.4 × 10-4 g/mL.
increased. We found that the pH in the SPS particle solutions varied gradually after its preparation: pH values of 2.0, 4.0, 7.0, 9.0, and 12.0 of the solutions as prepared changed to 2.0, 4.4, 6.5, 6.7, and 9.0, respectively, after one-half of a month, whereas the pH in the PSS solution, which dissolved molecularly, did not vary that much: a pH value of 9.0 changed to 7.0 and a pH value of 12.0 did not vary after one-half of a month of storage. These results imply that there are two factors influencing the pH: one is the absorption of carbon dioxide from air, which made the pH of 9.0 in both SPS and PSS solutions change to pH 6.7 and 7.0, respectively; another factor is that in the base media the protons inside the SPS particles released and moved gradually to aqueous bulk, neutralizing a part of hydroxyl leading to a pH decrease, which made the pH of 12.0 in SPS particles solution change to 9.0 but the pH of 12.0 in PSS solution did not change. Therefore, in basic medium, the local proton concentration inside the particles reduces, which makes the repulsive force of negatively charged sulfonated groups increase and the size of the particles increase. The higher pH is, the more evident the effect is. In addition, the size distribution of 32S-PS1 particles did not change significantly in the presence of 0.01 and 0.1 M NaCl at pH 2.0 (data not shown). Influence of DS on Dh Distribution of SPS Particles. Figure 3 shows the Dh for S-PS2 series with different DS ranging from 14 to 55 mol % at pH 2.0. The particle size decreases as DS increases, except for 36SPS2. As reported previously,6,7 for such surfactant-free particles, the average surface area stabilized by one hydrophilic moiety should be constant. More sulfonate groups can stabilize a larger surface area, leading to smaller particles. On the other hand, as DS increases, the
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Figure 4. Size distributions of the mixtures of 10 µM apo cyt c with different concentrations of 32S-PS1 at pH 2.0. The average Dh is about 175 nm for 32S-PS1 nanoparticles in the absence of apo cyt c (Figure 1).
average length of the hydrophobic polystyrene segments become shorter and more sulfonate groups are trapped in the particles. Thus, the particles could be swollen more by incorporating more water molecules. Therefore, the dependence of the particle size on DS would be the balance of these two factors. As a result, for S-PS2 series, Dh shows the maximum at DS being 36%. For S-PS1 series, similar results were obtained with a maximum Dh for 38S-PS1 (data not shown). DLS and AFM Studies on the Interaction of SPS Particles and Apo Cyt c. At pH 2.0, an apo cyt c molecule carries 24 positive charges and probably forms complexes with SPS particles through electrostatic interaction. Our previous study using circular dichroism demonstrated that apo cyt c underwent a conformational transition from random coil to R-helical structure on binding to negatively charged SPS particles.19 Figure 4 shows Dh distributions of the mixtures of 10 µM apo cyt c with different concentrations of 32S-PS1 at pH 2.0. A remarkable feature of the results is that the size and its distribution of 32S-PS1 particles (Figure 1) are completely changed. In the cases of very low SPS concentrations (curves a and b of Figure 4), where the positive charges of apo cyt c are in excess of the negative charges of SPS particles, there are two peaks in the Dh distributions and both of them are much larger in size than the parent SPS particles (Figure 1). Apo cyt c and SPS form complexes that have much larger Dh than the parent SPS particles. In addition, the excess of apo cyt c could link the complex particles forming large aggregates as particle size in the second peak, which is about 1 order of magnitude larger than SPS particles. When the SPS concentration increased to 7.0 × 10-5 g/mL (curve c), the mixture showed an even larger Dh and sedimentation was observed after 1 day of storage. In this case the ratio between the negative charges of 32S-PS1 and the positive charges of apo cyt c is 0.74, close to the stoichiometric value, so the mixture tends to form aggregate of the stoichiometric complex. Increasing 32SPS1 concentration to 1.4 × 10-4 g/mL (curve d), where the charges of SPS apparently are in excess of those of apo cyt c, f(Dh) shows one peak with a broad distribution ranging from 60 to 560 nm. It implies probably that the excess 32S-PS1 enriched on the periphery of the complex particles and prevented further aggregation. It is interesting to find that the complex particles in this composition are quite stable as a reproducible f(Dh) of the mixture was obtained when it was monitored over a period as long as 18 days. When the concentration of 32S-PS1 was increased further to 3.5 × 10-4 g/mL (curve e), two peaks appeared: one at 560 nm corresponds to the complexes,
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Figure 6. I1/I3 (solid symbol) and I338/I333 (hollow symbol) of pyrene as a function of 32S-PS1 concentration, pH 2.0.
Figure 5. AFM images of 32S-PS1 (A, 3 µm × 3 µm), the mixture of 1.4 × 10-4 g/mL of 32S-PS1 and 10 µM apo cyt c (B, 3 µm × 3 µm), and the mixture of 3.5 × 10-5 g/mL of 32SPS1 and 10 µM apo cyt c (C, 10 µm × 10 µm), pH 2.0.
while the other at 175 nm corresponds to the 32S-PS1 particles. This indicates the coexistence of the SPS particles and the complex particles in the solution. The AFM studies on 32S-PS1 particles (Figure 5A) show that the particles are circular and their diameter is about 80 nm. The diameter is significantly smaller than that measured by DLS (Figure 1). DLS provides the data for the particles swollen in solution, while AFM shows the image of dried particles on mica surface. Compared to Figure 5A, the image of the mixture of 1.4 × 10-4 g/mL of 32S-PS1 and 10 µM apo cyt c at pH 2.0 (Figure 5B), in which the negative charges of 32S-PS1 are in excess, shows two apparent differences. First, the smaller particles no longer display a regular circular outline; instead, apparent deformation is observed as a result of interaction of SPS particles with apo cyt c. Second, there is a coexistence of the larger aggregates and the primary particles. For the mixture of 3.5 × 10-5 g/mL of 32S-PS1 and 10 µM apo cyt c, where the positive charges are in excess, there are two kinds of large aggregates (Figure 5C, note the scale): one is about a few of hundreds of nanometers, and the other is about a few micrometers. The larger aggregates may be the assemblies of the smaller
ones linked by excess of apo cyt c. In short, the results of the AFM images shown in Figure 5B and 5C are qualitatively in agreement with the DLS results in curve d and curve b in Figure 4, respectively. Fluorescence Spectroscopy of SPS Particles. Pyrene has been widely used to monitor the association and micellization of macromolecules in solutions because its photophysical character changes when it transfers from a polar environment to a nonpolar one.6,26-28 Such changes include increases of both the quantum yield and the intensity ratio of the first to third bands (I1/I3) in its emission spectrum as well as a shift of the low-energy band of the La(S2fS0) transition from 333 to 338 nm in its excitation spectrum. Pyrene has a much lower solubility in water (about 10-7 M) than in hydrocarbons (0.075M); it significantly transfers into hydrophobic regions once the hydrophobic association occurs in aqueous solution. Figure 6 shows that the intensity ratio I1/I3 is about 1.7 when the 32S-PS1 concentration is extremely dilute, indicating that most of the pyrene molecules are surrounded by water molecules. When the concentration is higher than 3.5 × 10-6 g/mL, the ratio starts to decrease. A plateau value of 1.2 at a particle concentration higher than 1.4 × 10-4 g/mL indicates that most pyrene molecules transferred from water to a hydrophobic environment of the colloidal particles. The plateau value of I1/I3 is not as low as 0.95 measured in bulk polystyrene, indicating that some sulfonate groups and water molecules are incorporated in the particle cores. Furthermore, the increase of I338/I333 also indicates the formation of colloidal particles. At extremely dilute concentration of 32S-PS1, the ratio of I338/I333 is about 0.7, characteristic of pyrene in pure water. The turning point of the curve is similar to the intensity ratio of I1/I3. The plateau value of 1.3 at high 32S-PS1 concentration implies that most pyrene molecules transferred into the more hydrophobic environment of the colloidal particles. In addition, the fluorescence I1/I3 values of pyrene were 1.22, 1.21, and 1.21 in the presence of 0, 0.01, and 0.1 M NaCl, respectively, indicating that at such a low concentration the salt has no influence on the hydrophobic environment in the particles. As discussed above, our DLS studies showed that the SPS colloidal particles have formed at a concentration of SPS as low as 3.5 × 10-6 g/mL. However, at such a low concentration both I1/I3 and I338/I333 show typical values of hydrophilic media. This apparent discrepancy was reported previously, and the reason is that at such very (26) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039-2044. (27) Turro, N. J.; Lei, X.-G. Langmuir 1995, 11, 2525-2533. (28) Kagej, K.; Skerjanc, J. Langmuir 1999, 15, 4251-4258.
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Figure 7. SPS concentration dependence of I1/I3 (solid symbols) and I338/I333 (hollow symbols) of pyrene in S-PS2 particle dispersions, pH 2.0.
low concentrations the volume fraction of the formed particles is as low as 10-6 or 10-5 g/mL, so that the relative amount of pyrene transferred into the hydrophobic cores is not enough to make a significant change in I1/I3 and I338/I333.6,8 Although the fluorescence study could not be used to monitor the SPS particle formation, however, as we can see below, it provides useful information of the interaction of apo cyt c with SPS particles as I1/I3 and I338/I333 are good probes of the hydrophobicity of the medium where pyrene exists. Figure 7 shows the intensity ratio I1/I3 of pyrene versus S-PS2 concentrations with different DS. In the higher concentration range it can be clearly seen that generally the higher DS, the higher I1/I3, which is similar to the report of Essafi et al.9 These results indicate that with increasing DS, the particles show more hydrophilic and looser structure. Furthermore, the change in the intensity ratio I338/I333 for pyrene in excitation spectrum with different DS of SPS supports this explanation. Fluorescence Spectroscopy of the Interaction of SPS Particles and Apo Cyt c. Our previous investigation of the conformational transition of apo cyt c induced by SPS particles found that the folding could reach more than 90% of the R-helix content of native cyt c in solution. However, poly(sodium 4-styrenesulfonate) (PSS), which is 100% sulfonated polystyrene and soluble in water molecularly, induced only two-thirds of the R-helix content compared with SPS particles.19 It appears that the electrostatic interaction between PSS/SPS and apo cyt c induces an early partially folded state of apo cyt c, while the hydrophobic interaction between the nonpolar residues in apo cyt c and the hydrophobic cores of SPS particles extends the R-helix structure of apo cyt c, just like the folding of apo cyt c in lipid micelles. 10,11 To investigate the hydrophobic interaction between the cores of SPS particles and apo cyt c, the intensity ratio I1/I3 of pyrene in 19SPS1 or 38S-PS1 solution with or without apo cyt c at pH 2.0 was measured (Figure 8). The ratio I1/I3 of pyrene in 10 µM apo cyt c is 1.64, close to the ratio in water. However, the addition of apo cyt c into SPS particle solution caused a substantial change of I1/I3 of pyrene, an indication of the interaction of apo cyt c with SPS. At lower concentrations of SPS, both 19S-PS1-apo cyt c and 38S-PS1-apo cyt c complexes show much lower I1/I3 than the corresponding SPS particles. This can be due to two factors: first, the positive charges of apo cyt c neutralize the negative charges of 19S-PS1 and 38S-PS1 particles, leading to more hydrophobic microdomains in the solution, and second, the volume fraction of the SPS particles is much larger when they are combined with the added apo cyt c. At a
Figure 8. Dependence of I1/I3 ratio of pyrene on 19S-PS1 or 38S-PS1 concentration (lower X axis) and the ratio of the concentration of 19S-PS1 or 38S-PS1 to apo cyt c (mol/mol) (upper X axis) at pH 2.0, with or without 10 µM apo cyt c. The I1/I3 for 10 µM apo cyt c is 1.64 in the absence of the SPS nanoparticles.
high SPS concentration range, the 19S-PS1-apo cyt c complex particles are more hydrophilic than 19S-PS1 particles, as indicated by the I1/I3 differences. It may be explained that apo cyt c inserts into the cores of 19S-PS1 particles, which disrupts the original hydrophobic structure of 19S-PS1 particles, leading to a more hydrophilic environment. Compared with 19S-PS1, 38S-PS1 has more sulfate groups both inside and on the surface of the particles, and hence, the I1/I3 of pyrene in 38S-PS1 particles shows more hydrophilicity over the whole concentration range. Apo cyt c may neutralize the negative charges of the sulfate groups on the surface and/or inside 38S-PS1 particles so that the complex particles are still more hydrophobic than 38S-PS1 particles at high SPS concentration range. Mixtures of 10 µM apo cyt c with different concentrations of SPS at pH 2.0 showed that a significant helical structure appeared when the concentration of SPS was 3.5 × 10-5 g/mL, and the helical structure increased as SPS concentration increased.19 The studies in this report show that at very low concentration, SPS can still interact with apo cyt c, although SPS cannot induce a significant conformational transformation of apo cyt c at pH 2.0. At pH 7.0, the I1/I3 of pyrene in 19S-PS1 is 1.22 at the high SPS concentration range (Figure 9), larger than that at pH 2.0, indicating that the 19S-PS1 particles are more hydrophilic at pH 7.0. Combined with the results of DLS, the size of SPS particles is larger at pH 7.0 than the size at pH 2.0 (Figure 2); a conclusion can be made that the structure of SPS particles is looser because of the stretching of the chains with negatively charged sulfonate groups at higher pH. Over the whole 19S-PS1 concentration, addition of apo cyt c causes a decrease of I1/I3 of pyrene; however, the difference is minor, which is due to the fact that apo cyt c carries much less positive charges at pH 7.0 compared to pH 2.0; thus, it causes weaker interaction at pH 7.0. Studies of the conformational transition showed that the helical structure content of apo cyt c induced by SPS particles was less at pH 7.0 than that at pH 2.0,19 which can also be explained by the weaker interaction between apo cyt c and SPS at pH 7.0. At the high 19S-PS1 concentration range, the I1/I3 of pyrene in the mixtures at pH 7.0 is still smaller than that in the parent SPS particles, inconsistent with 19S-PS1-apo cyt
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Figure 9. Dependence of I1/I3 ratio of pyrene on 19S-PS1 concentration (lower X axis) and the ratio of the concentration of 19S-PS1 to apo cyt c (mol/mol) (upper X axis) at pH 7.0, with or without 10 µM apo cyt c. The ratio I1/I3 for apo cyt c is 1.82 in the absence of the SPS nanoparticles.
c at pH 2.0 but similar to 38S-PS1-apo cyt c at pH 2.0. There are also two explanations: (1) the hydrophobic microdomains are formed because of the interaction of apo cyt c and the sulfate groups on the surface of 19SPS1 particles and (2) apo cyt c still inserts into the cores of 19S-PS1 particles neutralizing the negative charges of sulfate groups. Previous results showed that poly(sodium 4-styrenesulfonate) induced only two-thirds of the R-helix content compared with SPS particles,19 implying that the cores of SPS particles play an important role in the interaction of apo cyt c and SPS particles. Furthermore, the structure of 19S-PS1 is looser at pH 7.0 as mentioned above, which may help apo cyt c insert into the particles.
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Conclusion When SPS in THF/H2O (50/50, v/v) was added dropwise into an excessive amount of water, the stable colloidal particles formed because the hydrophobic backbone chains collapsed and the ionic groups transferred toward the particle surfaces. The size distribution of SPS particles is independent of the SPS concentration but increases as the pH in the solution increases. A low concentration of NaCl has no significant influence on the size distribution of SPS. Apo cyt c carries positive charges in pH < 10.5 aqueous solution and can form complexes with SPS. The size and structure of the SPS-apo cyt c complex particles are dependent on the SPS/apo cyt c compositions. When SPS is in excess, apo cyt c interacts with SPS particles, forming stable complexes, and excessive SPS particles bind to the periphery of the complexes, preventing further aggregation. When apo cyt c is in excess, apo cyt c links the complexes, forming much larger particles. The fluorescence study demonstrates that apo cyt c not only can neutralize negative charges on the surface of SPS particles, but may also insert into the cores, disrupting the original structure of SPS particles. Combining our previous result that SPS particles can induce apo cyt c from random coil to helical structure19 and the present results, we conclude that the interaction of SPS particles and apo cyt c is similar to the interaction of lipid micelles and apo cyt c.10,11 Perhaps sulfonated polystyrene particles can partially mimic a lipid membrane environment. Acknowledgment. The financial support of the National Natural Science Foundation of China (NSFC Project 20074006) and the Foundation for University Key Teacher by the Ministry of Education of China are gratefully acknowledged. LA035844L
