Aggregation Behavior of Triple Helical Polysaccharide with Low

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J. Phys. Chem. B 2010, 114, 4945–4954

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Aggregation Behavior of Triple Helical Polysaccharide with Low Molecular Weight in Diluted Aqueous Solution Yangyang Zhang, Sheng Li, and Lina Zhang* Department of Chemistry, Wuhan UniVersity, Wuhan 430072, China ReceiVed: October 20, 2009; ReVised Manuscript ReceiVed: March 6, 2010

It has been proven in our previous work that the lentinan triple helical chains with high weight-average molecular weight (Mw ) 1.71 × 106) formed easily a self-entangle one, and no ordered aggregates were detected. In the present work, we used the ultrasonic method to degrade the lentinan triple helical chains to obtain a sample with a mean value of Mw ≈ 5.0 × 105 g/mol. Subsequently, its dilute aqueous solution properties were studied by dynamic light scattering (DLS). The relaxation time distributions exhibited two modes (fast and slow) with different relaxation time scales. The fast mode was attributed to the relaxation of an individual triple helical lentinan, whereas the slow mode indicated the formation of large aggregates. On the basis of the scattering wave vector dependencies for the scattering intensity and for the amplitudes and characteristic times associated with the relaxation modes, the molecular parameters were calculated by combing static LS with DLS. The values of the radius of gyration for individuals (〈Rg〉indi) and aggregates (〈Rg〉agg) were 48.2 and 75.4 nm, and those of the hydrodynamic radius for individuals (〈Rh〉indi) and aggregates (〈Rh〉agg) were 14.9 and 98.4 nm, respectively. Furthermore, the structure-sensitive dimensionless parameter of individuals (Findi ) 3.23) and aggregates (Fagg ) 0.766) indicated that the individual triple helical chains were stiff, whereas the aggregates existed as compact clusters. The aggregates consisted of short triple helical chains by packing close to form “faggot-like” assembly, and the ordered aggregates (near 10%) coexisted with the predominant triple helical chain in the aqueous solution. Atomic force microscopy provided straightforward evidence on the shape of the triple helical chains and their aggregates in water. Introduction Lentinan is a neutral polysaccharide extracted from the fruiting body of Lentinus edodes, consisting of a β-(1f3)-Dglucose residues, to which there are two β-(1f6)-D-glucosyl residues for every five main-chain glucose residues.1,2 There is abundant evidence that lentinan can promote human health by stimulating the immune system via activating immune cells such as lymphocytes, macrophages, DCs, NK cells, Th cells, Tc cells, and B cells, and this stimulation has been used to treat various cancers and AIDS.3-5 It is noted that only lentinan having a moderate molecular weight and a triple helical conformation exhibits significant bioactivities.6 It has been confirmed in our previous work that lentinan exists as triple helical chains in aqueous solution with morphologies of worm-like linear, circular, and crossover as a result of the intrachain entanglement, whereas it changes to single flexible chains in dimethyl sulfoxide (DMSO).7 The triple helical structure of lentinan is sustained by the intra- and intermolecular hydrogen bonds, and it exhibits high stability over a wide range of temperature. However, the disentanglement and dissociation of the triple helical chains appear at elevated temperature. The thermally induced conformational transition from triple helix to single coil has occurred at about 130-145 °C.8 Moreover, the diversity of the hydrogen bonds leads to the multiple conformation of the triple helical polysaccharide in the DMSO/water solutions with a change of temperature.9 Interestingly, the denatured lentinan (singleflexible chains) can be renatured to triple helical conformation by dialyzing against water,10 and its collapse coil chains can * To whom correspondence should be addressed. Phone: 86-2787219274. Fax: 86-27-68762005. E-mail: [email protected].

very easy form large aggregates.11 These facts have proven that the strong hydrogen-bond interaction exists in lentinan. β-(1f3)-D-glucan with triple helical structures, such as schizophyllan and scleroglucan, could assemble to be new triple helices with polynucleotides through the hydrogen-bonding and hydrophobic interactions.12,13 The polysaccharide-ploynucleotide complex is an excellent carrier for immunostimulatory CpGDNAs.14,15 Scleroglucan can form triple helical trimers (aggregates) in aqueous solutions,16 whereas schizophyllan triple helices with a moderate molecular weight can align parallel to each other, leading to the cholesteric liquid crystal behavior.17 However, no ordered aggregates and liquid crystal behaviors for lentinan were observed in our previous work. Why did the ordered aggregates not appear? Did these aggregations occur in special conditions, and if they did, what were the conditions and what kind of aggregates could form? These interesting questions have inspired us to look at the aggregation behaviors of triple helical lentinan in water. Hydrogen bonding, as one of the essential noncovalent forces, plays an important role not only in physical aggregates but also in the processes of self-assembly.18 Classical examples are the essential action for complementary base-pairing in DNA19 and the formation of supramolecular helices.20 Many testing techniques and computational models have been used to investigate the hydrogenbonding-induced self-assembly behaviors, such as NMR spectroscopy,21,22 molecular kinetics calculation,23,24 and various spectra.25,26 Particularly, dynamic light scattering (DLS) can provide information to give insight into the influence of the architecture and the dynamic progress on aggregation and self-assembly of polymers. DLS is a sensitive and reliable method to investigate aggregation behavior of polymer solutions. The individual polymer chains can

10.1021/jp9100398  2010 American Chemical Society Published on Web 03/29/2010

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be described as a fast mode, whereas aggregates show a slow diffusion. Thus, it would offer an alternative interpretation of the bimodal DLS spectrum. An extremely slow diffusive mode in lowsalt poly(L-lysine) solutions by DLS was proposed in 1978.27,28 Since then, a lot of effort has been devoted to the investigation of the phenomenon in many different systems, including low-ionicstrength DNA solutions,29,30 associating polymer solutions,31 linear neutral polymer systems in good solvents,32 and nearly all classes of polyelectrolyte solutions.33-37 For interpretations of the slow mode, the most common one should be that it is a result of the scattering from aggregates or clusters formed by multipolymer chain domains. The lentinan triple helical chains with relatively high molecular weight form easily a self-entangle one.8 It is not hard to imagine that the self-entanglement of the triple helical chains would increase the steric hindrance to prevent the forming of ordered aggregates. In the present work, we used an ultrasonic method to degrade the long chains of lentinan; this did not destroy their triple helical conformation.9 The dilute solution properties and the molecular parameters of triple helical lentinan with low molecular weight were studied by static light scattering (SLS) and DLS to clarify the aggregates and supermolecular structure. Atomic force microscopy was used to further confirm the existence of individual helical chains and aggregates as well as their sizes and shape. We hope to provide significant information on the aggregation and selfassembly behavior of triple helical polysaccharides with low molecular weight in water.

Zhang et al.

q ) |b q| )

4πn0 θ sin λ0 2

()

(1)

where n is the refractive index of the solvent (1.332 at 25 °C and 1.326 at 70 °C for the water), λ0 is the wavelength of light in the vacuum, and θ is the scattering angle. In our experiments, the scattering angle θ has been varied between 30 and 150°, which corresponds to scattering wave vectors q in the range of 6.8 × 10-3 to 2.6 × 10-2 nm-1. Atomic Force Microscope. To observe atomic force microscopy (AFM), lentinan was dissolved in deionized water by drastically stirring for 24 h and dialyzed with deionized water for 5 days. The polysaccharide solutions were filtered through a 0.22 µm filter (NYL, 13 mm Syringe filter, Whatman Inc. U.S.A.) and diluted with deionized water to the polymer concentration of 15 µg/mL. A 10 µL drop was deposited onto freshly cleaved mica and allowed to dry in air for 3 h at room temperature. The specimen was examined using a Picoscan AFM (Molecular Imaging, Tempe, AZ, U.S.A.) in a MAC mode with commercial MAC lever II tips (Molecular Imaging, U.S.A.), with a spring constant of 0.95 N/m. A piezoelectric scanner with a range up to 6 µm was used for the image. The scanner was calibrated in the xy directions using a 1.0 µm grafting and in the z direction using several conventional height standards. The measurement was performed in air at ambient pressure and 25 °C, and the image was stored as 256 × 256 point arrays. Data Analysis

Experimental Section Sample Preparation. Lentinan was isolated from fruiting bodies of Lentinus edodes cultivated in Suizhou of China by extraction with 1.25 M NaOH/0.05% NaBH4. The detailed procedure of extraction has been reported previously.8 To obtain the sample with low molecular weight, lentinan was dissolved in distilled water with a concentration of 1.5 mg/mL and exposed to 20-25 kHz ultrasonic irradiation by an ultrasonic cell disruptor (JY92-IID, Ningbo Scientz Biotechnology Co., Ltd., China) in an ice water bath for 120 min. Sonicated solutions were purified by using the reprecipitation method via acetone at room temperature. The precipitates were dissolved in water again to obtain clear solutions, then dialyzed against distilled water for 5 days, filtered, and finally lyophilized to give the colorless flakes, and coded as LFS-120. The intrinsic viscosity ([η]) of LFS-120 in water was measured by viscometry and was calculated from Huggins and Kraemer equations to be 223 mL/g, which was much lower than that of the original one. This indicated that the resulting lentinan sample (LFS-120) did possess low molecular weight. Polysaccharide solutions were prepared by mixing the required amounts of LFS-120 and distilled water in a flask by stirring gently for 4 h at 50 °C, and then, they were kept stirring overnight at room temperature. The mass concentration c (mg/ mL) was in the range from 0.25 to 3.0 mg/mL. For lightscattering experiments, all of the solutions were filtered directly into the light-scattering cells through a 0.22 µm pore size filter (NYL, 13 mm Syringe filter, Whatman, Inc., U.S.A.). Light Scattering Measurements. In all of the light-scattering measurements, we used an ALV/DLS/SLS-5000E light-scattering goniometer (ALV/CGS-8F, ALV, Germany) with vertically polarized incident light of wavelength 632.8 nm from a He-Ne laser equipped with an ALV/LSE-5003 light-scattering electronics and multiple tau digital correlator. In static LS, the excess of scattered intensity I(q) with respect to the solvent was measured, and the magnitude of the scattering wave vector q is given by

Static Light Scattering. Corrections to the absolute timeaveraged scattering intensities Rvv(q) (excess Rayleigh ratio) were made using a toluene sample reference, for which the excess Rayleigh ratio is well-known as follows

Rvv(q) )

( )

〈Isolution(q)〉 - 〈Isolvent(q)〉 nsolvent Rref 〈Iref(q)〉 nref

2

(2)

Rvv(q) of a dilute polymer solution at concentration c (g/mL) is related to the weight-average molecular weight (Mw). The radius of gyration (Rg) and the scattering wave vector (q) are expressed as38

Kc 1 1 1 + 〈R2g〉zq2 + 2A2c ≈ Rvv(q) Mw 3

(

)

(3)

The scattering constant is K ) 4π2n2(dn/dc)2/NAλ04, where dn/dc is the refractive index increment, NA is Avogadro’s number, and A2 is the second virial coefficient. The value of dn/dc for the lentinan polysaccharide in water was determined to be 0.140. The plots of [Kc/Rvv(q)]cf0 versus q2 and [Kc/ Rvv(q)]qf0 versus c lead to 〈Rg2〉 and A2, respectively. Dynamic Light Scattering. In DLS measurements, the normalized autocorrelation function g(2)(t) of scattered light intensity was measured, and it can be expressed in terms of the field autocorrelation function or, equivalently, in terms of the autocorrelation function of the concentration fluctuations g(1)(q,t) (i.e., the time autocorrelation function of the scattered electric field) through39

g(2)(q, t) )

〈I(q, 0)I(q, t)〉 ) A + β|g(1)(q, t)| 2 〈I(q, 0)〉2

(4)

Aggregation Behavior of Triple Helical Polysaccharide

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where A is the measured baseline and β is the coherent factor that depends on the detection optics. For a broadly distributed relaxation, |g(1)(q,t)| is related to a characteristic relaxation time distribution (G(Γ)) as

|g(1)(q, t)| )

∫0

∞

() Γ q2

[

]

with c f 0

if qRg , 1

(10)

Results and Discussion

G(Γ) exp(-Γt)dΓ

(5)

On the basis of eqs 4 and 5, G(Γ) can be obtained from the Laplace inversion of each measured g(2)(q,t) value by using a CONTIN program.40 From the Stokes-Einstein equation, it is convenient to calculate the hydrodynamic radius Rh as41

Dc)0 )

c 1 c 1 + q2(R2g) ) I(q, c) I(0, c) 3

q)0,c)0

)

kBT 6πηsRh

(6)

where Dc)0 is the translational diffusion coefficient D at indefinite dilution, kB is the Boltzmann constant, T is the absolute temperature, and ηs is the solvent viscosity. In this study, both fast and slow relaxation modes were observed, so that the time autocorrelation function of the scattered electric field can be described by a sum of two relaxations42 〈Efast(q, t)E* fast(q, t' + t)〉 + 〈Eslow(q, t)E* slow(q, t' + t)〉 〈I(q)〉 g(1)(q, t) ) Afast(q) exp(-Γfastt) + Aslow(q) exp(-Γslowt)

g(1)(q, t) )

Fast Mode and Slow Mode of Lentinan in Dilute Solutions. DLS experiments of the triple helical lentinan LFS-120 in water were done to clarify the applicability of the fast mode and slow one in this system. The value of the estimated “overlap concentration” c* ()4.5 mg/mL) for the lentinan sample was deduced from the reciprocal of the intrinsic viscosity [η]-1. All of the polymer concentrations were below c*, which acts as a demarcation value of the dilute and semidilute regimes. Figure 1a shows two DLS autocorrelation functions and the result of the CONTIN analysis for two LFS-120/water solutions with concentrations of 0.25 and 3 mg/mL (scattering angle θ ) 90°, temperature T ) 25 °C). There were two overlapping peaks in the hydrodynamic time distributions, indicating the existence of two modes (fast and slow) with different relaxation time scales. The fast mode can be attributed to normal molecules, namely, an individual triple helical chain of lentinan, whereas the slow mode represents their aggregates. At c ) 0.25 mg/ mL, a smaller intensity peak for the slow mode was observed, whereas it shifted to high τ at c ) 3.0 mg/mL. This suggested that the aggregation increased with an increase of the polymer concentration. Figure 1b shows the scattering vector dependence of intensity-intensity time correlation functions of the LFS-

(7) where Efast(q) and Eslow(q) are the fast and the slow electric scattered fields, respectively, and are fluctuating independently. Γfast and Γslow are the fast and the slow relaxation times, respectively. Afast(q) and Aslow(q) are the corresponding amplitudes, where Afast(q) + Aslow(q) ) 1. Previously, G(2)(q,t) was analyzed with different methods,43 including a combination of two exponential functions.36,44,45 By using a double-exponential fit or a CONTIN fit results and the Stokes-Einstein equation, each measured G(2)(τ) leads to two average hydrodynamic radii 〈Rh〉fast and 〈Rh〉slow. Combination of Static and Dynamic Light Scatterings. The corresponding amplitudes of the two modes measured by DLS can be used to analyze the static LS data. The scattering intensity from each mode can be calculated from the total timeaveraged scattering intensity (〈I(q)〉) measured in static LS and A(q) from DLS by using a double-exponential fit or a CONTIN fit. Ifast(q) and Islow(q) are defined as the time-average intensities associated with the fluctuations of polymer concentration with corresponding short and long relaxation times and

Ifast(q) ) Afast(q)I(q) Islow(q) ) Aslow(q)I(q)

(8)

I(q) ) Itotal(q) ) Ifast(q) + Islow(q)

(9)

For infinite dilution solutions, the plots of c/I(q,c) versus q2 can be extrapolated to q f 0 to give intercepts c/I(0,c). If the length scale q-1 is sufficiently large compared to Rg of the polymers, the average value of 〈Rg2〉 can be determined from the intercept and the initial slope of these plots using a scattering inverse Lorentzian law of the form39

Figure 1. (a)Typical autocorrelation function of LFS-120 in water as measured by DLS at c ) 3 (O) and 0.25 mg/mL (3) at 25 °C and the CONTIN inverse Laplace transforms of the DLS correlation functions corresponding to the hydrodynamic time distributions; open dots are a CONTIN analysis (scattering angle θ ) 90°). (b) Scattering vector dependence of intensity-intensity time correlation functions of LFS120 in water with c ) 1.5 mg/mL at 25 °C, where the delay time (τ) is scaled with q2. The inset shows the corresponding hydrodynamic radius distributions calculated from CONTIN analysis.

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Figure 2. The corresponding hydrodynamic radius distributions of LFS-120 in water with c ) 0.75 mg/mL at different storage times (scattering angle θ ) 90°), where the corresponding hydrodynamic radius distributions were calculated from CONTIN analysis.

120/water solution with a concentration of 1.5 mg/mL at 25 °C. By using the Stokes-Einstein equation, we could calculate a hydrodynamic radius (〈Rh〉) and its distribution f(Rh), as well as the values from each G(D). The inset of Figure 1b shows the corresponding apparent average hydrodynamic radius (〈Rh〉app) and their distributions f(Rh,app) calculated from the CONTIN analysis. A certain extent of similarity appeared in the curves of f(Rh) at different scattering angles, suggesting that the distributions have regularity in all scattering angles. The calculated 〈Rh〉app values for individual chains ranged from 15 to 25 nm, whereas those for the aggregates were in the range from 100 to 200 nm. This indicated that the aggregation of triple helical lentinan occurred in the aqueous solution. Figure 2 shows the 〈Rh〉app distributions of LFS-120 in water at different storage times at θ ) 90 °C (CONTIN analysis). The curve of f(Rh) changed slightly after the polysaccharide solution was filtered and stored at 25 °C for 30 h. However, with an increase of the storage time from 30 h to a week, the curve of f(Rh) hardly changed. This suggested that this system, where individual lentinan chains coexisted with their aggregates, was in equilibrium and at least had temporal stability for more than a week. In order to minimize the influence of storage time for different polysaccharide solutions, all of the experiments were carried out after the solutions were filtered through 0.22 µm filters and stored at 25 °C for 48 h. To further confirm the presence of two dynamically separable particles in LFS-120 aqueous solution, we changed the measurement temperature to study the 〈Rh〉app values. Figure 3 shows the 〈Rh〉app distributions of LFS-120 in water at different temperatures at θ ) 90° (CONTIN analysis). At 25 °C, the 〈Rh〉app distributions of LFS-120 with a concentration of 0.6 mg/ mL exhibited a shoulder peak, which corresponded to the fragments of triple helical chains (shorter chains). Interestingly, the shoulder peak disappeared at 40 and 70 °C. The results indicated that an increase of temperature led to a decrease of the triple helical chain fragments. With an increase of temperature, the two overlapping peaks became sharper and separate, but the peak position of either individuals or aggregates hardly changed on the whole. At 70 °C, the two peaks respectively corresponding to the individual triple helical chains and their aggregates were well-separated. Thus, T ) 70 °C was selected as the measurement temperature for further studies. Typical scattering profiles for 2.4 mg/mL LFS-120 in water are shown in Figure 4. The area (A) under each peak is directly related to the scattering intensity from each relaxation mode.

Zhang et al.

Figure 3. The corresponding hydrodynamic radius distributions of LFS-120 in water with c ) 0.6 mg/mL at different temperatures (scattering angle θ ) 90°), where the corresponding hydrodynamic radius distributions were calculated from CONTIN analysis and shifted by A.

Figure 4. Scattering vector dependence of intensity-intensity time correlation functions of LFS-120 in water with c ) 2.4 × 10-3 g/mL at 70 °C, where the corresponding hydrodynamic radius distributions were calculated from CONTIN analysis.

The corresponding amplitudes are dependent on the scattering wave vector q. Figure 5a shows the q dependence of the Aslow/ Afast ratio for the LFS-120 solutions at concentrations of c ) 0.6 and 3 mg/mL at 70 °C. The Aslow/Afast ratio increased with an increase of the scattering angle for both concentrations, and a similar phenomenon was observed in other concentrations. The variation of Aslow/Afast at q ) 0 with the LFS-120 concentration is shown in Figure 5b. The concentration independence of Aslow/Afast indicated a continuous increase of the concentration for the individuals. According to Buhler’s results on polyelectrolyte polysaccharides,46,47 if the aggregation process follows a closed association mechanism, there is a critical aggregation concentration (cac). When the concentration of polysaccharide is higher than the cac, the concentration of individuals is expected to saturate and to be equal to the critical aggregation concentration (cac < c*). It means that the area (A) related to the scattering intensity of the fast mode would be independent of the total polysaccharide concentration, and the variation of Aslow/Afast at q ) 0 would increase as the polysaccharide concentration increases. However, we have no evidence for the existence of such a cac in the present case. The difference of concentration dependence of Afast between LFS-120 and the theoretical models of polyelectrolyte could be explained by the fact that the aggregate mechanism of triple helical chain lentinan was significantly different from that of the polyelectrolyte. Figure 6a shows the autocorrelation function at θ ) 90 and 30° for LFS-120 with concentrations of 0.6 and 3 mg/mL,

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Figure 5. (a) Scattering wave vector dependence (a) of Aslow/Afast for LFS-120 solutions at c ) 3 and 0.6 mg/mL; the line represents the fit of the data. (b) LFS-120 solution concentration dependence of Aslow/ Afast at q ) 0.

Figure 6. (a) Typical autocorrelation function LFS-120 in water with c ) 0.6 mg/mL (scattering angle θ ) 90°) and c ) 3 mg/mL (θ ) 40°). The dots represent the experimental data, the dashed line show single-exponential functions, and the solid line show double-exponential functions. (b) Relationship between 1/〈τ〉 and q2 calculated using the data measured at 70 °C shown in (a); c ) 0.6 (open symbols) and 3 mg/mL (filled symbols).

respectively. On the basis of the peak shape of the hydrodynamic radius distribution f(Rh) from CONTIN analysis, we assumed two relaxation processes (fast and slow) in the letinan solution, as we predicted. The single-exponential fit was obviously dissatisfactory, whereas a double-exponential function was successful to fit the data at any θ, any LFS-120 concentration, and any temperature in our study. From the fitted results, we calculated the decay time, 〈τ〉, at each scattering angle. The relationship between 1/〈τ〉 and q2 for the two concentrations measured at 70 °C is shown in Figure 6b. For two relaxation modes of fast and slow, a linear relationship was obtained. To minimize the fitting error, we used both the CONTIN Laplace inversion and double-exponential methods to analyze each time correlation function. The 〈τ〉 value is defined as the peak top value in the decay time distribution in CONTIN analysis. Both of them can be calculated to obtain the average characteristic decay times (〈τ(q)〉fast and 〈τ(q)〉slow), respectively, for the fast and slow modes. As shown in Figure 6b, the 〈τ〉 values calculated from a double-exponential fit were close to that from a CONTIN analysis. Moreover, the relaxation time of the individuals was significantly faster than that of the aggregates, indicating a good applicability of two modes in the lentinan aqueous system. Figures 7 shows the dynamic Zimm plot of 1/〈τ(q)〉fast (a) and 1/〈τ(q)〉slow (b). They condense both the concentration and angular dependence on a single grid.47 It was noted that two fitting methods led to a slight difference in the shape of the Zimm plot, but the plot of 1/〈τ(q)〉 versus q2 was a straight line passing through the origin. This indicated that both the fast and the slow relaxations are diffusive. The two slopes reflected, respectively, the translational diffusion coefficients 〈D〉fast and 〈D〉slow. From

the 〈D〉fast and 〈D〉slow data, we estimated the average dynamic correlation length of both the fast mode and the slow mode. Figure 8 shows the comparison of the concentration dependence of 〈Rh〉fast and 〈Rh〉slow calculated from CONTIN Laplace inversion and double-exponential methods. The 〈Rh〉fast values were almost independent of the LFS-120 concentration, even though the values calculated from the former were a bit larger than those of the latter. As expected, the 〈Rh〉slow values increased with an increase of the polysaccharide concentration. It could be explained that in a more concentrated solution, the chance of defective triple helical chains meeting each other was more than that in a relatively dilute one. The extrapolation of the line to zero concentration of LFS-120 in Figure 7 gave the 〈Rh〉fast value of 16.9 nm in CONTIN analysis and 14.9 nm in doubleexponential analysis. For the slow mode, the 〈Rh〉slow value was calculated to be 99.6 and 98.4 nm in CONTIN analysis and double-exponential analysis at zero polysaccharide concentration, respectively. In view of the above results, 〈Rh〉fast of individual triple helical chains was about 16 nm, and the 〈Rh〉slow value of their aggregates was 99 nm. Molecular Parameters from Fast and Slow Modes. The traditional way to analyze SLS data is to use the Zimm plot. However, the normal Zimm plot is unable to give accurate values of triple helical lentinan solutions. For this special complex system, the individuals coexist with aggregates in the aqueous solution, but a usual Zimm plot can only provide the information of the polymer mixture. On the basis of eq 3, from a typical Zimm48 plot of dilute solutions of LFS-120 in water at 70 °C (SFigure 1a, Supporting Information), the extrapolation of [[Kc/Rθ]cf0,qf0 led to Mw ) 1.20 × 106 g/mol, and the slopes

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Figure 7. Scattering vector (q2) and polymer concentration dependence of the average characteristic decay time (〈τ(q)〉) of the fast mode (upper) and the slow mode (lower) calculated from two different fittings of measured [g(2)(τ,q) - 1] [CONTIN (open circles) and double exponential (solid circles)].

Figure 8. The comparison of 〈Rh〉fast and 〈Rh〉fast calculated from CONTIN Laplace inversion (open symbols) and double-exponential methods (solid symbols) as a function of polymer concentration (c).

of [Kc/Rθ]cf0 versus q2 and [Kc/Rθ]qf0 versus c gave 〈Rg〉 ) 63.4 nm and A2 ) 3.9 × 10-8 mol cm3/g2, respectively. Compared to the result of the Zimm plot measured at 25 °C (SFigure 1b, Supporting Information), the Mw value exhibited a remarkable decrease from 1.87 × 106 to 1.20 × 106 g/mol, but 〈Rg〉 diminished only 6 nm, and the value of A2 at 70 °C almost was the same as that at 25 °C. The positive but small values of A2 suggested that water was close to the θ solvent for LFS-120 at either 25 or 70 °C. Moreover, the existence of aggregates in the polymer solutions often leads to a very small or even negative value of A2. The overlap concentration (c*) estimated from Mw/(〈Rg〉3NA)53 was 9.1 mg/mL at 25 °C and 7.3 mg/mL at 70 °C. It was further confirmed that all of the concentrations used here were within the dilute solution range.

Zhang et al.

Figure 9. Scattering vector (q2) dependence of average excess scattering intensity related to the fast mode (〈I(q)fast〉) (a) and the slow mode (〈I(q)slow〉) (b) of 3.0 mg/mL LFS-120 in water at 70 °C. Data of the fast and slow modes were calculated by combining SLS and DLS measurements from two different fittings [CONTIN (open circles) and double exponential (solid circles)]. The details are presented in the text.

These results gave powerful support that there was indeed some aggregates coexisting with individual chains in the lentinan aqueous solutions. By using a fast and slow mode, the aggregates could be well-distinguished from individuals. From the Zimm plot, the angular dependence of the total scattered intensity, which represents the summation of the scattered intensity due to the individuals and the aggregates, can be given. The fraction of the scattered intensity corresponding to the two different species could be estimated as the ratio of the peak areas based on the CONTIN analysis and the exponent result of double-exponential analysis. Therefore, the scattered intensity resulting from the individuals and the aggregates at each scattering angle can be separated and estimated, as shown in Figure 9a. As expected, 〈I(q)〉fast was independent of the scattering angle because of its short correlation length and the stiffness of the triple helix chains. Figure 9b shows the scattering angle dependence of both 〈I0〉fast/〈I(q)〉fast and 〈I0〉slow/〈I(q)〉slow of the lentinan solution with a concentration of 3 mg/mL. The intensity ratio 〈I0〉fast/〈I(q)〉fast associated with the fast mode was almost independent of wave vector, therefore; it is impossible to determine Rg by using the Lorentzian law (eq 10). On the other hand, from the intercept and the initial slope, each plot of 〈I0〉slow/〈I(q)〉slow versus q2 can be used to obtain a radius of apparent gyration 〈Rg〉slow, which is related to the slow mode at each concentrantion. Figure 10 summarizes the concentration dependence of different average radii, 〈Rh〉 and 〈Rg〉. By combing static LS with DLS analyzed by both the CONTIN method and double-exponential analysis, we got two

Aggregation Behavior of Triple Helical Polysaccharide

J. Phys. Chem. B, Vol. 114, No. 15, 2010 4951 TABLE 1: Molecular Characteristics Results of LFS-120 in Water Solution at 70°C Mw × 10-4 (g mol-1) 〈Rg〉z (nm) 〈Rh〉 (nm) 〈Rg〉z/〈Rh〉 total individuals aggregates

120.0a 46.1b 763.0b 717.9d

63.4a 48.2b 75.4b 69.8c 71.3d

14.9b 98.4b

3.23 0.766 0.709 0.725

a

Data from regular Zimm plot. b Data from separated Zimm plot by combining SLS and DLS results. c Data calculated from Lorentzian law. d Data calculated from eqs 11 and 12.

Figure 10. Concentration dependence of different average radii 〈Rh〉 and 〈Rg〉 of LFS-120 in water solutions.

groups of 〈Rg〉slow. The 〈Rg〉slow values were independent of the polysaccharide concentration, and the difference between the two analysis results was tiny. From the extrapolation of the line to zero concentration of LFS-120, the values of 〈Rg〉slow were calculated to be 69.3 nm using the CONTIN method and 71.3 nm using double-exponential analysis. The two values were close to each other. This indicated that double-exponential analysis was an applicable model for our polysaccharide system. The separated Zimm plots could be used to calculate molecular parameters and the exact content of aggregates in lentinan dilute aqueous solutions. On the basis of eq 2 and the scattered intensity calculated from the above data, Rvv(q) at every scattering angle for both the individuals and the aggregates can be estimated at each concentration. The concentration ratio of the individuals and the aggregates at each polymer concentration should be determined first, so that we can obtain the information such as Mw of the individuals and the aggregates from the Zimm plot. Although we cannot obtain the exact concentration for the individuals directly through experiment, it should be much larger than that of the aggregates since, over the concentration range studied (0.55-3.1 mg/mL), they yields two intensity modes of comparable strength but quite different sizes. A same and successful assumption has been made in the laser light scattering study of a rigid-rod polyelectrolyte.49 In addition, from the concentration independence of the variation of Aslow/Afast at q ) 0, we can assume that although the ratio f (f ) xfast/(xfast+ xslow) ) cfast/(cfast + cslow) ) cfast/ctotal) is not a constant at different polymer concentrations, the difference can be quite small, as shown in Figure 4. In view of the above analysis, we attempted to assume individuals as predominant species. For example, the individuals were about 90 wt % of the total polysaccharide concentration over the concentration range used, and meanwhile, the other 10 wt % was due to the aggregates. To investigate the influence of the individuals percentage on Mw, we changed the value of f (f ) cfast/ctotal) from 0.84 to 0.99, and the corresponding molecular weigh changed from 4.35 × 105 to 5.13 × 105 g/mol (The detail information was shown in STable 1, Supporting Information). This little change of Mw further confirmed our assumption that the amount of the individuals was much larger than that of the aggregates. Zimm plots of the fast and slowly diffusing fractions (f ) 0.89) are shown in SFigure 2 (Supporting Information). It showed appreciable dispersion in the data. Simultaneous fitting to all of the data points in the Zimm procedure helped to reduce this noise, thereby yielding the Mw and Rg values for the two populations of molecules. The results are summarized in Table 1. The Mw values were 4.61 × 105 g/mol for the individuals and 7.63 ×

106 g/mol for the aggregates. The 〈Rg〉z values were found to be 48.2 and 75.4 nm for the population of fast and slowly diffusing molecules, respectively. Therefore, in the dilute aqueous solution, the content of individual triple helical lentinan was nearly 90%, and their aggregates were only 10 wt %. Comparison between Experiment and Calculation. On the basis of the calculated data and our analysis, the individuals may have the same shape and sizes, whereas the aggregates have different sizes and shape within the diluted solution range. Wu et al. have deduced equations to establish the compositions of Mw, 〈Rg〉z in a complex polymer system. If only individuals and aggregates coexist in solutions at a given temperature, Mw, 〈Rg〉z may be expressed by50

Mw,app ) fMw,indi + (1 - f)Mw,agg

(11)

〈Rg〉z,appMw,app ) f〈Rg〉z,indiMw,indi + (1 - f)〈Rg〉z,aggMw,agg (12) Here, f denotes the weight fraction of individuals, as mentioned before. In view of the results of Mw,app ) 1.20 × 106 g/mol and 〈Rg〉z,app ) 63.4 nm from the Zimm polt, there are only two equations and five unknown parameters, f, Mw,indi, Mw,agg, 〈Rg〉z,indi, and 〈Rg〉z,agg, of the individuals and the aggregates. Considering the small difference of the molecular weight of individuals caused by changing the value of f, we used a Mw,indi of the fast mode obtained from the Zimm plot as a known parameter, getting a value of Mw,indi ) 4.61 × 105 g/mol as f ) 0.89 in eq 11; the calculated value of Mw,agg was 7.17 × 106 g/mol. From a typical Zimm plot, the slopes of [Kc/Rθ]cf0 versus q2 and [Kc/Rθ]qf0 versus c led to 〈Rg〉, which was independent of concentration. Thus, we could use any value of Rg,indi and Rg,agg as a known parameter without leading to obvious error. On the basis of the description above, when making Rg,indi ) 48.2 nm fixed, a value of Rg,agg ) 71.3 nm was easily calculated according to eq 12 (data in Table 1). The calculated Mw,agg and 〈Rg〉z,agg values were in good agreement with the data from the Zimm plot of the slow mode (Mw,agg ) 7.63 × 106 g/mol, 〈Rg〉z,agg ) 75.4 nm). This strongly confirmed that by combing LLS with DLS, plotting two separated Zimm plots was a credible and simple way to analyze data for this system, where both individuals and aggregates coexisted. Conformation Description of Individuals and Aggregates. It is well-known that the radius of gyration (〈Rg〉) is defined as the mean distance between the segment and the mass center, and it reflects the chain occupied space. The hydrodynamic radius (〈Rg〉) is regarded as a quantity to characterize the dimension of macromolecules in solution, taking into account the hydrodynamic interaction. Additional structural information about the individuals and aggregates can be obtained by calculating the ratio of the two radii, namely, F ) Rg/Rh; F is a

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Zhang et al.

Figure 11. (a) MAC mode AFM topographic images of lentinan aqueous solution at 25 °C. (b) The 3D image of (a). (c) The 3D image of triple helical chains within a small scale.

structure-sensitive dimensionless parameter that can be used as a qualitative determination of the main macromolecular architecture in the solution. The F values expected for different chain conformations have been reported.51,52 For a uniform and nondraining sphere, F is about 0.77, and for an extended rigid chain (or worm-like one), F is more than 2.0. Especially, for a triple helical polysaccharide having side chains attached to every third unit, such as scleroglucan, F ) 2.42.53 By using the 〈Rg〉fast and 〈Rh〉fast values in our findings, F for the individual triple helical letinan in the dilute aqueous solution could be estimated to be ∼3.23, indicating an extended stiff rod. Interestingly, the F values corresponding to the aggregates were in the range from 0.709 to 0.766 calculated by different analysis methods. The data for the aggregates were comparable to that for a uniform and nondraining sphere (∼0.77). This suggested that triple helical chains close packed to each other and formed tight clusters or ordered aggregates. Was it random coil-like or ordered clusters by parallel accumulation of triple helical chains? To clarify the shape of the aggregates, we analyzed the data in Table 1 in detail. When triple helical chains of lentinan gathered into aggregates, the molecular weight increased significantly from 4.61 × 105 to 7.17 × 106 g/mol; however, the value of 〈Rg〉 exhibited only a slight increase (from 48.2 to 71.3 nm). It is not hard to imagine that triple helical chains parallel aligned to each other and close packed to form “faggot-like” clusters, leading to the slight change of 〈Rg〉 as Mw increased considerably. The similar aggregate behavior of triple helical PPG with low molecular weight has been reported, showing that it formed high ordered triple helical “fasces-like” clusters, a microfibrillar superstructure.54 No ordered aggregates were observed in our previous lentinan aqueous solution because the self-entanglement of triple helical chains having high molecular weight could prevent the formation of the ordered aggregates.8 In the present work, more fragments of triple helical chains existed after the ultrasonic treatment, as shown in Figure 2. A shoulder peak in the

hydrodynamic radius distributions of lentinan with a concentration of 0.6 mg/mL proved the existence of these short chains and segments. The short chains existed as rods and showed a strong trend to form aggregates; therefore, the short triple helical chains and fragments aggregated to form clusters more easily, compared to long chain lentinan. It also could explaine why schizophyllan triple helices having decreased molecular weight could align parallel to form a liquid crystal17 and display as anisotropic fluids. To further confirm that the aggregates close packed to form “faggot-like” clusters in the letinan aqueous solution, AFM was used to observe their shape. Moreover, the size of the chains and their aggregates were calculated. Figure 11a shows the MAC mode AFM images of the lentinan sample in deionized water. As expected, there were big aggregates in the AFM topography with a height of 15-34 nm. To observe the detailed topography of the individual triple helical chains of lentinan, a smaller region without aggregates was chosen. As shown in Figure 11b, the AFM topography revealed the presence of predominantly linear patterns along with some circular and crossover species. The average height of the individual triple helical chains was about 1.6 nm; this value is similar to the previously reported mean thickness of triple helical polysaccharides.8,55 In tapping mode AFM with soft materials, the measured height does not exactly correspond to the true height and is usually less56,57 due to the deformation of the soft surface by the AFM tip. The results from AFM revealed that the predominant species of the LFS120 sample in aqueous solution existed as a worm-like chain with different length, circular, and crossover species, whereas the aggregates only account for a small part. This was good support to the results calculated from light-scattering studies. The contour length (L) of the triple helical lentinan can be calculated from the molar mass per unit contour length (ML ) 2200 nm-1).7,9 According to the definition for a worm-like chain, the mean L value of individual triple helical chain 〈L〉indi ()Mw/ML) for the lentinan sample in water was estimated to

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Figure 12. Schematic of triple helical lentinan in water solution. Stiff rods represent the individuals, and the spheres represent aggregates of single chains.

be 210 nm, which is comparable with that observed in the AFM image (170 nm). As shown in Figure 11a and b, the aggregates exhibited a hill-look pattern. It confirmed directly that the short chains of triple helical lentinan with different lengths packed parallel to form ordered “faggot-like” aggregates. Interestingly, their maximum height was 35 nm, and the average height and diameter were about 24 and 40 nm, respectively. The persistence length (Lp) reflects the stiffness of polymer chains, and the value of Lp for lentinan with triple helical conformation is about 120 nm.7,9 The large Lp value of lentinan indicated that the short chains were stiff enough to keep as “straight rods” to form ordered “faggot-like” aggregates. On the basis of the results of aggregates measured from the AFM image, an approximate 〈L〉indi,short of short triple helical chains was calculated to be 24 nm, suggesting that numerous shorter chains packed to form the aggregates. Obviously, 〈L〉indi,short was far shorter than 〈L〉indi, which further confirmed that only those short chains could form “faggot-like” aggregate clusters in triple helical lentian aqueous solutions. Moreover, the apparent mean aggregation number 〈Nagg〉 can be calculated using Mw,indi and Mw,agg by

Nagg )

Mw,agg Mw,indi

(13)

where Mw,indi is the weight-average molecular weight of individual triple helical chains of lentinan and Mw,agg is the weight-average molecular weight of their aggregates. The Nagg value was given to be 16 ( 1, which was much lower than that obtained from AFM. It further supported that the predominant species to form aggregates in water was very short chains with high rigidity. On the basis of the above results, a schematic diagram to describe the chain conformation of lentinan with a relatively small molecular weight (Mw ≈ 5.0 × 105 g/mol) in water was proposed in Figure 12. The triple helical lentinan chains and their aggregates coexisted in water; the predominant species was individual triple helical chains, whereas their aggregates were few. The short chain or fragments of triple helical lentinan formed “faggot-like” clusters coexisting with individuals in the aqueous solution.

Conclusion The time autocorrelation function of the scattered electric field measured by dynamic light scattering was used successfully to detect the bimodal peaks corresponding to individual triple helical chains and their aggregates in the lentinan aqueous system. A combination of static and dynamic LLS results created their molecular characteristic parameters of both the fast mode and the slow one, which correspond to the relaxation of individual triple helical lentinan and their physical associations. By using fast and slow modes we successfully described the chain conformation of triple helical lentinan in dilute aqueous solution, indicating coexistence of the predominant triple helical chain and few aggregates. The results from DLS and AFM revealed that the short triple helical chains parallel aligned to each other and close packed to form ordered “faggot-like” clusters. Only the rod-like chains of lentinan having low molecular weight could form ordered aggregates as a result of the strongest interaction and the smallest steric hindrance between each chain. This result also explained why schizophyllan triple helices having decreased molecular weight could align parallel to form a liquid crystal.17 The formation of such ordered aggregates associated with short triple helical chains provided important information on the three-dimensional structure of the polysaccharides. Acknowledgment. We gratefully acknowledge the National Basic Research Program of China (973 Program, 2010CB732203), the major grant of the National Natural Science Foundation of China (30530850), the National Natural Science Foundation (20404010 and 20874078), and the High-Technology Research and Development Program of China (2006AA02Z102). Supporting Information Available: Additional Zimm plots and a comparison of molecular weights and the ratio of gyration. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Sasaki, T.; Takasuka, N. Carbohydr. Res. 1976, 47, 99–104. (2) Zhang, P.; Zhang, L.; Cheng, S. Biosci. Biotechnol. Biochem. 1999, 63, 1197–1202.

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(3) Jong, S. C.; Birmingham, J. M. AdV. Appl. Microbiol. 1992, 37, 101–134. (4) Brauer, D.; Kimmons, T.; Phillips, M. J. Agric. Food Chem. 2002, 50, 5333–5337. (5) Moradali, M.; Mostafavi, H.; Ghods, S. Int. Immunopharmacol. 2007, 7, 701–724. (6) Zhang, L.; Li, X.; Xu, X.; Zeng, F. Carbohydr. Res. 2005, 340, 1515. (7) Zhang, L.; Zhang, X.; Zhou, Q.; Zhang, M.; Li, X. Polym. J. 2001, 33, 317–321. (8) Wang, X.; Xu, X.; Zhang, L. J. Phys. Chem. B 2008, 112, 10343– 10351. (9) Wang, X.; Zhang, Y.; Zhang, L. J. Phys. Chem. B 2009, 113, 9915– 9923. (10) Zhang, X.; Zhang, L.; Xu, X. Biopolymers 2004, 75, 187–195. (11) Xu, X.; Zhang, X.; Zhang, L.; Wu, C. Biomacromolecules 2004, 5, 1893–1898. (12) Sakurai, K.; Mizu, M.; Shinkai, S. Biomacromolecules 2001, 2, 641–650. (13) Sakurai, K.; Shinkai, S. J. Am. Chem. Soc. 2000, 122, 4520–4521. (14) Mizu, M.; Koumoto, K.; Anada, T.; Matsumoto, T.; Numata, M.; Shinkai, S.; Nagasaki, T.; Sakurai, K. J. Am. Chem. Soc. 2004, 126, 8372– 8373. (15) Takeda, Y.; Shimada, N.; Kaneko, K.; Shinkai, S.; Sakurai, K. Biomacromolecules 2007, 8, 1178–1186. (16) Yanaki, T.; Norisuye, T. Polym. J. 1983, 15, 389. (17) Chen, W.; Takahiro, S; Akio, T. Macromolecules 1999, 32, 1549– 1553. (18) Feng, T.; Bernasek, S. L. J. Am. Chem. Soc. 2005, 127, 12750– 12751. (19) Keitaro, Y.; Seiichi, N.; Masakazu, M.; Norio, T. J. Am. Chem. Soc. 2003, 125, 8982–8983. (20) Tatiana, G.; Stefano, L.; Paolo, M. J. Am. Chem. Soc. 2003, 125, 14741–14749. (21) Manasi, P.; Bhate, J. C.; Manish, A. M. J. Am. Chem. Soc. 2009, 2009, 9579–9589. (22) Ken, C. F.; Fraser, S. J. Am. Chem. Soc. 2006, 128, 10707–10715. (23) Kenneth, E. M.; Eric, G; Thierry, M; James, D. W. J. Am. Chem. Soc. 2007, 129, 4306–4322. (24) Daniel, J.; Mandell, I. C. J. Am. Chem. Soc. 2007, 129, 820–827. (25) Dong, H.; Sergey, E.; Paramonov, L. J. Am. Chem. Soc. 2007, 129, 12468–12472. (26) Shiki, Y.; Shun, K.; Yoshihiro, K. J. Am. Chem. Soc. 2009, 131, 5408–5410. (27) Lin, S. C.; Lee, W. I.; Schurr, J. M. Biopolymers 1978, 17, 1041. (28) Mathiez, P.; Mouttet, C.; Weisbuch, G. Biopolymers 1981, 20, 2381.

Zhang et al. (29) Newman, J.; Tracy, J.; Pecora, R. Macromolecules 1994, 27, 6808– 6811. (30) Borsali, R.; Nguyen, H.; Pecora, R. Macromolecules 1998, 31, 1548–1555. (31) Klucker, R.; Munch, J. P.; Schosseler, F. Macromolecules 1997, 30, 3839. (32) Buhler, E.; Munch, J. P.; Candau, S. J. J. Phys. II 1995, 5, 765. (33) Tanahatoe, J. J.; Kuil, M. E. J. Phys. Chem. B 1997, 101, 9233. (34) Forster, S.; Schmidt, M.; Antonietti, M. Polymer 1990, 31, 781– 792. (35) Sedlak, M. Physical Chemistry of Polyelectrolytes; Radeva, T., Ed.; Marcel Dekker, Inc: New York, 2001; pp 1-58. (36) Sedlak, M. J. Chem. Phys. 1997, 107, 10805. (37) Sedlak, M. Langmuir. 1999, 15, 4045–4051. (38) Wu, C.; Siddiq, M.; Woo, K. F. Macromolecules. 1995, 28, 4914– 4919. (39) Cummins, H. Z.; Pike, E. R. Photon Correlation and Light Beating Spectroscopy; Plenum Press: New York—London, 1974; pp 9–41. (40) Provencher, S. W. J. Chem. Phys. 1978, 69, 4273. (41) Brochard, F.; de Gennes, P. G. Macromolecules 1977, 10, 1157. (42) Rinaudo, M. Macromolecules 2000, 33, 2098–2106. (43) Ngai, T.; Wu, C.; Chen, Y. J. Phys. Chem. B 2004, 108, 5532. (44) Esquenet, C.; Buhler, E. Macromolecules 2002, 35, 3708–3716. (45) Onuma, K.; Kubota, T.; Tanaka, S. J. Phys. Chem. B 2002, 106, 4318–4324. (46) Esquenet, C.; Buhler, E. Macromolecules 2001, 34, 5287–5294. (47) Jin, F.; Chi, W. J. Phys. Chem. B 2007, 111, 2255–2261. (48) Teraoka, I. Polymer Solution; John Wiley & Sons, Inc: New York, 2002. (49) Liu, T. B.; Rulkens, R. Macromolecules 1998, 31, 6119–6128. (50) Wu, C.; Siddiq, M. Macromolecules 1995, 28, 4914–4919. (51) Burchard, W. AdV. Polym. Sci. 1999, 143, 113–194. (52) Burchard, W. Theory of cyclic macromolecules. In Cyclic polymers; Semlyen, J. A., Ed.; Elsevier Applied Science Publishers: London and New York, 1986; pp 43-84. (53) Sletmoen, M.; Geissler, E.; Stokke, B. T. Biomacromolecules 2006, 7, 858–865. (54) Gawronski, M.; Park, J. T.; Magee, A. S.; Conrad, H. Biopolymers 1999, 50, 569–578. (55) Theresa, M.; McIntire; David, A. B. J. Am. Chem. Soc. 1998, 120, 6909–6919. (56) Hansma, H. G. J. Vac. Sci. Technol., B 1996, 14, 1390–1394. (57) Tamayo, J.; Garacia, R. Langmuir 1996, 12, 4430–4435.

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