Determination of the Triple Helical Chain Conformation of β-Glucan by

Jan 8, 2014 - Triple helical polysaccharides (t-polysaccharides) are easily gelated in water, resulting in difficult fractionation, leading to the com...
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Determination of the Triple Helical Chain Conformation of β‑Glucan by Facile and Reliable Triple-Detector Size Exclusion Chromatography Sheng Li, Yao Huang, Sen Wang, Xiaojuan Xu, and Lina Zhang* Department of Chemistry, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: Triple helical polysaccharides (t-polysaccharides) are easily gelated in water, resulting in difficult fractionation, leading to the complex and time-consuming chain conformational characterization. Moreover, the fractionation is not always successful due to the coexistence of individual chains and aggregates. In this work, we developed a facile and reliable method to rapidly and accurately characterize the chain conformation of t-polysaccharide without fractionation needed in traditional conformation characterization. A triple helical β-1,3-glucan (t-β-1,3-glucan), extracted from the fruiting bodies of Lentinus edodes, was identified to consist of a β-1,3-glucan with two β-1,6-D-glucopyranoside branchings for every five β-1,3-glucopyranoside linear linkages by one- and two-dimensional NMR and GC-MS analysis. The chain conformations of the t-β-glucan in aqueous solution and in DMSO were successfully characterized by a combination of size exclusion chromatography (SEC), multiangle static light scattering, a differential refractometer, and a capillary viscosity detector (triple-detector SEC). The results revealed that the predominate species of the t-β-glucan in a 0.15 M NaCl aqueous solution existed as a triple helical conformation with high chain stiffness, and a few aggregates (4%) coexisted here. The Mark−Houwink and ⟨S2⟩1/2 versus Mw equations of individual triple helical chains and aggregates were obtained simultaneously, and the results confirmed again the coexistence of two kinds of chain conformations. The fractal dimension indicated that the aggregate in the aqueous solution was a kind of reversible microgel with a 3D network structure. Furthermore, the chain morphology of the t-βglucan in aqueous solution was observed directly by transmission electron microscopy and atomic force microscopy to support the worm-like chain for the individuals and 3D network for the aggregates. The triple-detector SEC technology was facile and reliable for the system with two fractions of different chain conformation, and the test time required was only 1/30 of what the traditional method needed. gyration ⟨S2⟩z1/2) and intrinsic viscosity ([η]) data. The whole process is complicated and time-consuming (usually half of a year). Moreover, the traditional fractionation is not always successful to obtain samples with different molecular weights and narrow distributions due to aggregation. Thus, the result is usually not trustworthy due to the coexistence of the individual chains and the aggregates.15 It is noted that the molecular weight and chain conformation data are very important for understanding the function and role of polysaccharides in life. It has been found that the bioactivity of lentinan is greatly affected by its chain conformation and molecular weight.16 The βglucan with triple helical chain conformation shows higher antitumor activity, and it would decrease significantly or even disappear when the β-glucan chain was denatured into the random coils. Thus, a basic understanding of the chain conformation is essential for the successful interpretation of

1. INTRODUCTION In recent years, polysaccharides with triple helical chain conformation were found to play an important role in the life process and treatment of diseases. Lentinan, extracted from Lentinus edodes, is a kind of β-1,3-glucan with two β-1,6-Dglucopyranoside branchings for every five β-1,3-glucopyranoside linear linkages.1,2 Since the discovery of antitumor activity of lentinan by Chihara,3,4 research on the bioactivity of lentinan has attracted much attention.5 Besides the antitumor activity, lentinan also exhibits other bioactivities such as antivirus,6 antiinflammation,7 and activation of human immune system function8 as a kind of immune activator. In our laboratory, lentinan has been demonstrated to exhibit triple helical chain conformation in water9 and to be denatured into a random coil under some extreme conditions, such as in dimethylsulfoxide (DMSO),10,11 in NaOH aqueous solution (>0.08 M),12,13 or at high temperature (>135 °C).14 To determine the triple chain conformation, purification and fractionation of the polysaccharide must be carried out for a long time. After fractionation of the polysaccharide, each fraction should be characterized to obtain the molecular weight (Mw), root-mean-square radius of © 2014 American Chemical Society

Received: August 31, 2013 Revised: January 7, 2014 Published: January 8, 2014 668

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Perkin-Elmer Co.) in the region of 400−4000 cm−1. The samples were prepared by using the KBr disk method. 1H NMR, 13C NMR, and heteronuclear correlation spectra between a carbon atom and the hydrogen atom connected directly (HMQC) to the t-β-glucan in DMSO-d6 were obtained on a Varian INOVA-600 spectrometer in the proton noise-decoupling mode with a standard 5 mm probe at 25 °C. The sample concentration was about 5 wt %. The chemical shifts were referred to the signals of tetramethylsilane (TMS). GC-MS analysis was carried out on a GCMS-QP2010 Plus (Shimadzu, Japan) GC-MS machine equipped with a capillary injection system and a FTD detector. The capillary column used in the test was an Rxi-5MS (30 m × 0.32 mm ×0.25 μm). The test temperature ranged from 150 to 220 °C with a heating rate at 2 °C/min, and the detector temperature was 280 °C. The carrying gas was N2. 2.3. Determination of Chain Conformation. The Mw, ⟨S2⟩z1/2, and [η] values of the β-glucan in 0.15 M NaCl and in DMSO were measured on the SEC columns by combination with MALS (DAWN HELLEOS-II, equipped with He−Ne laser, λ = 663.4 nm), RI (Opitilab T-rEX, λ = 658.0 nm), and Vis (ViscoStar-II). The SEC columns used in the test were Shodex-OHpak SB-806 M HQ (8.0 mm × 300 mm) for 0.15 M NaCl and Shodex-GPC KF-806 L (8.0 mm × 300 mm) for DMSO. The temperature of both columns was kept at 40 °C, and the flowing rate was 0.6 mL/min. Astra software was used to collect the data. All of the measurements were finished within less than 1 week, which was much faster than the traditional method. TEM images were observed on a JEOL JEM-2010 (HT) electron microscope, using an accelerating voltage of 200 kV. A drop of the t-β-glucan aqueous solution (0.5 mg/mL) was placed on Cu grids precoated with carbon films, and the sample was coated with carbon to improve the contrast for the low contrast of polysaccharide chains. For the AFM observation, the t-β-glucan solution was filtered through a 0.45 μm filter (NYL, 13 mm syringe filter, Whatman Inc., U.S.A.) and diluted with deionized water to the polymer concentrations of 5 and 20 μg/mL. A 10 μL drop was placed on freshly cleaved mica and allowed to dry in air. The AFM observation was carried out on a Picoscan atomic force microscope (Molecular Imaging, Tempe, AZ) in the MAC mode with commercial MAC lever II tips (Molecular Imaging, Tempe, AZ), with a spring constant of 0.95 N/m. The obtained image was stored as 256 × 256 point arrays.

the correlation of structure to bioactivities of polysaccharides and their application in the biomedical field. Since the establishment of the hot water extraction method by Chihara first,3,4 an improved method created by Yap and Ng17 has been more wildly used in the extraction of lentinan. In our lab, a kind of green, efficient, and low-cost method has been established, and the resultant lentinan sample is easily dissolved in water.13 Although the chain conformation and bioactivities of the lentinan sample have been studied widely in our previous work, some problems such as characterization of the chain conformation for polysaccharides with complex structure always puzzled us. Meanwhile, a lot of research is blocked by the absence of enough proof for the triple chain structure, especially the coexistence of triple helical chains and the aggregates in lentinan aqueous solution, which makes it difficult to determine the accurate molecular weight and chain structure of lentinan. Additionally, polysaccharides with triple helical conformation usually form some complex structures in the denaturation−renaturation process, just like cyclization or supercoiling structures, which had been well-studied in former reports.18−20 All of these problems make polysaccharide characterization much more difficult. A facile and reliable determination of chain conformation and Mw of polysaccharides is essential for further research and product development. In this work, we combined the size exclusion chromatography (SEC) with multiangle static light scattering (MALS), a differential refractometer (RI), and a capillary viscosity detector (Vis) to collect the distribution of molecular weights (Mw) and the gyration of radius (⟨S2⟩1/2) and intrinsic viscosities ([η]) of polysaccharides simultaneously. Moreover, each data point constituting the distribution curves can be regarded as one fraction of polysaccharides to obtain a set of data including Mw, ⟨S2⟩1/2, and [η]. Therefore, it is a feasible pathway to obtain the molecular parameters of individual chains and aggregates and to determine the chain conformations of both components. We thus tried to establish such a method with triple-detector SEC tech to determine the chain conformations of both the individual chain and aggregates in the β-glucan aqueous solution. Additionally, transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to directly observe the chain morphology and size for confirming the result obtained from the newly established method.

2. EXPERIMENTAL SECTION 2.1. Materials and Sample Preparation. β-Glucan was extracted from the fruit bodies of Lentinus edodes (purchased from the supermarket) via the method established in our lab, and the detailed process has been reported elsewhere.13 After the primary purification and lyophilization, the β-glucan sample with a shape of colorless flakes was obtained. To distinguish it from the general β-glucan, the sample was denoted as t-βglucan. The yield of the β-glucan was up to 5%. Other chemical regents such as sodium hydride (NaH), methyl iodide (CH3I), sodium borohydride (NaBH4), and DMSO were purchased from Shanghai Chemical Regent and were used directly. 2.2. Characterization of Chemical Structure. To determine the monomer units and the linkage sequence, the t-β-glucan was first methylated according to the method described in the literature with some improvements.21 Then, the reaction product was hydrolyzed and was acetylated. The final production was analyzed by GC-MS. The Fourier transform infrared (FT-IR) spectrum of the t-βglucan was recorded on an FT-IR spectrometer (model 1600,

3. RESULTS AND DISCUSSIONS 3.1. Chemical Structure of t-β-Glucan. The chemical structure of the t-β-glucan was analyzed initially through the FT-IR result. As shown in Figure S1 (Supporting Information), the absorption peaks were assigned to a stretching vibration of the hydroxyl group (3400 cm−1), a symmetric stretching vibration of the pyran ring, an in-plane deformation vibration of the hydroxyl group (1460 cm−1), a shear stretching vibration of CH2 (1420 cm−1), a deformation stretching vibration of CH (1370 cm−1), and a stretching vibration of C−O (1000−1200 cm−1). The anomeric absorption peak at 890 cm−1, indicated by the red arrow in Figure S1 (Supporting Information) and the absence of a peak in the range of 1650 to 1700 cm−1, indicated an existence of neutral β-configuration polysaccharide.22 To further determine the monomer units and linkage sequence, the t-β-glucan was methylated, hydrolyzed, and acetylated. The composition of the final product was analyzed 669

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between hydrogen atoms, the 1 H NMR spectrum of polysaccharides is difficult to resolve, and the 1H NMR spectrum of β-glucan has been rarely reported before. Figure S3 (Supporting Information) shows the HMQC spectrum of the β-glucan in DMSO-d6. The HMQC spectrum can reflect the coupling information between the carbon atom and the hydrogen atom connected directly, and the 1H NMR signals can be resolved according to the assignment of corresponding carbon atoms. The marks beside the cross peaks indicate the corresponding C−H groups. The assignment results are summarized in Table 2. From the analysis of the above results, the t-β-glucan sample extracted from Lentinus edodes was a β1,3-glucan with two β-1,6-D-glucopyranoside branchings for every five β-1,3-glucopyranoside linear linkages. Figure 2 shows the chemical structure model of the t-β-glucan repeating unit.

by GC-MS, as shown in Figure 1. The results of the GC-MS profile are listed in Table 1. The t-β-glucan consisted of three

Figure 1. GC-MS profile of the methylated β-glucan.

Table 1. GC-MS Results of the Methylated β-Glucan peak

time (s)

percentage (%)

2,3,4,6-tetra-O-methylglucitol 2,4,6-tri-O-methylglucitol 2,4-di-O-methylgucitol

593 748 977

31.8 43.6 24.6

kinds of repeating units, including 2,3,4,6-tetra-O-methylglucose (1-O), 2,4,6-tri-O-methylglucose (1,3-O), and 2,4-di-Omethylglucose (1,3,6-O). The content ratio was calculated to be 1.2:1.8:1, indicating that two β-1,6-glucose residues existed as branches for every five β-1,3-glucose residues in the backbones. The 13C NMR spectrum of the t-β-glucan in DMSO-d6 (Figure S2, Supporting Information) showed that all chemical shift peaks were totally in agreement with the β-1,3-D-glucan. The peak at δ = 103.7 ppm represented the anomeric carbon (C1) of β-glucan. Usually, the anomeric signal of polysaccharides with β-configuration is higher than δ = 100 ppm, and the one with α-configuration is lower than 100 ppm.23 Moreover, no peak appeared in the range of δ = 160−200 ppm, proving that the β-glucan was a kind of neutral polysaccharide with β-configuration, which was consistent with the FT-IR result. Compared with the 13C NMR spectrum reported previously and by using the automatic determination method designed by Widmalm,24 the signals were assigned, and the data are listed in Table 2. The glycosidic linkages of the glucan containing (1 → 3)-linked and (1 → 6)-linked β-Dglucopyranosyl were confirmed by the presence of 3-Osubstituted signal at δ = 87.0/77.3, and O-substituted-CH2-6 signal at δ = 69.1. Due to the presence of mutual coupling

Figure 2. The chemical structure model of the t-β-glucan repeating unit. Red: O atoms; gray: C atoms; white: H atoms.

3.2. Chain Conformation of the t-β-Glucan. The Mw, ⟨S2⟩z1/2, and [η] values of the polysaccharide in aqueous solution can be obtained online by the method at the same time. Especially, when the individual triple helical chains and aggregates coexist in the polymer solution as two different components, the SEC−LLS−RI−Vis combination can distinguish the molecular weight and chain conformation of the two fractions. The conformation of the t-β-glucan in 0.15 M NaCl was characterized by triple-detector SEC. Figure 3 shows the Mw versus retention time plots and the SEC chromatograms recorded by the LLS detector and RI detector. Clearly, Mw did not always change linearly with the retention time. Near the

Table 2. 13C NMR and 1H NMR Chemical Shift of the βGlucan chemical shift (ppm) atoms C1/C1′ C-2/C-2′ C-3/C-3′ C-4/C-4′ C-5/C-5′ C-6/C-6′

13

1

103.7 73.5/74.4 87.0/77.3 69.1/70.7 76.9/75.3 61.6/69.1

4.2 2.94; 3.0 3.12 3.06; 3.18 3.0 3.6; 3.2

C NMR

H NMR

Figure 3. Mw versus retention time plots (Mw, green) as well as the chromatograms recorded by the MALS detector (LS, red) and RI detector (RI, blue) for the t-β-glucan in a 0.15 M NaCl aqueous solution. The peak area was divided into the aggregation region (I) and the individual triple helical chain region (II). 670

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upper size exclusion limit, a similar nonlinear dependence of Mw on the retention time is often observed.25 Concerning the high upper size exclusion limit of the column (>2 × 107) used here, it is suggested that different size exclusion effects existed in the system, rather than the phenomenon caused by the column limitation. According to the linear relationship, two areas were obtained, as shown in Figure 3. In the range of 2 × 105 to 2 × 106 (denoted as area II), Mw linearly decreased with increasing retention time, which corresponded to the individual t-β-glucan. When the Mw value was higher than 2 × 106 (denoted as area I), the Mw versu retention time dependence displayed a curve, which was ascribed to aggregation of t-βglucan, leading to a sharp increase in molecular weight at low retention time. According to the differential distribution curve (the blue curve) as shown in Figure 3, the individual and aggregate parts were calculated to be 96 and 4%, respectively. Thus, the fractions with different Mw ranges were chosen to analyze the molecular parameters of the β-glucan. As a result, the average values of Mw, Mn, ⟨S2⟩z1/2, and [η] of the fractions in areas I and II were calculated and are listed in Table 3. As

⟨S2⟩z

Mw × 10−5

Mn × 10−5

⟨S2⟩z1/2 (nm)

[η] (g/mL)

t-β-glucan (area I) aggregation (area II) apparent average (area I + II)

9.6 28.5 10.8

7.1 28.0 7.5

99 153 109

576 800 612

= 1.81M w 0.30

(2 × 106 < M w < 2 × 107)

⟨S2⟩z

1/2

for aggregates

(1)

for individuals

(2)

= 1.01 × 10−2M w 0.71

(2 × 105 < M w < 2 × 106)

Usually, the exponent (α) of polymers with the aggregates is about 0.3, while the one with a flexible chain conformation in good solvent is between 0.5 and 0.6, and the one with the rigid chain conformation is larger than 0.6.26 According to the power law theory,27 in area I, the α value of 0.30 indicated the aggregates, whereas α = 0.71 for area II revealed the rigid chain conformation of the t-β-glucan in the aqueous solution. Therefore, the fraction in area II mainly consisted of rigid chains, while a few aggregates coexisted in the aqueous solution. According to the Kratky−Porod worm-like cylinder model28 and Yamakawa−Yoshizaki theory,29,30 the molar mass per unit contour length (ML) and persistence length (q) can be calculated from the linear fitting between (Mw2/12⟨S2⟩)2/3 and Mw.31 To determine the chain conformation of the individual t-β-glucan, the data in area II were used to calculate the molecular parameters. Figure 5 shows the (Mw2/12⟨S2⟩)2/3

Table 3. Molecular Characteristic Results of the t-β-Glucan in a 0.15 M NaCl Aqueous Solution for the Fractions in Both Areas sample

1/2

shown in Figure 3, the data were collected at equal interval retention times, and finally, all of the data constituted the curve (the red or the blue). On the basis of regarding each data point as an individual fraction, the relationships of ⟨S2⟩z1/2 versus Mw and [η] versus Mw could be obtained. Figure 4 shows the Figure 5. (Mw2/12⟨S2⟩)2/3 versus Mw plots for the t-β-glucan in 0.15 M NaCl and the linear fitting curve (dashed line).

versus Mw plots for the β-glucan sample in 0.15 M NaCl and the linear fitting curve. The ML (=2170 nm−1) and q (=80 nm) were obtained from the intercept and slope of the fitting curve, respectively. Figure 6 shows the experimental data and the theoretical curve of ⟨S2⟩z1/2 = kMwα calculated from the triple helical model.32,33 The experimental curves coincide well with

Figure 4. ⟨S2⟩1/2 versus Mw polts for the t-β-glucan sample in 0.15 M NaCl and the linear fitting curves (dashed line).

⟨S2⟩z1/2 versus Mw plots and the linear fitting curves as the function of ⟨S2⟩z1/2 = kMwα. As we expected, two linear curves with clearly different slopes were observed, indicting two kinds of components with different conformations. The two equations for the t-β-glucan sample in 0.15 M NaCl (lentinan concentration < 0.1 mg/mL) from the linear fitting were as follows

Figure 6. Comparison between the measured ⟨S2⟩ z1/2 for the t-βglucan in 0.15 M NaCl and the theoretical values calculated for different q with ML fixed to 2170 nm−1. 671

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the theoretical model when q = 80 nm. It can be concluded that the β-glucan (area II, Mw < 2 × 106) existed as a triple helical chain conformation in the aqueous solution. By using the [η] data of vast fractions of the t-β-glucan collected by the capillary viscosity detector in the SEM pattern, the Mark−Houwink equation can be established directly without a complicated fractionation process. Figure 7 shows

could be explained as the t-β-glucan existing as stiff triple helical chains in the aqueous solution and easily forming aggregates through the formation of the intermolecular hydrogen bonds. The microgels were reversible, which increased with an increase of the polymer concentration and disaggregated into individual triple helical chains at ultralow concentration. The chain morphology of polysaccharides in aqueous solution can be observed directly by TEM or AFM measurements. Because NaCl would precipitate out from the salt solution and the glucan chain conformation was nonsensitive to the existence of salt, as reported previously, the morphology observation of the t-β-glucan chain by TEM and AFM was carried out by dissolving the polysaccharidesolution in water.24,43,44 Considering the composition (mainly as C, H, O) and the ultrasmall size of polysaccharides, it is difficult to observe the morphology of the polysaccharide chain under TEM observation. Usually, the TEM observation of macromolecules with light elements (such as DNA, protein, polysaccharides, etc.) was carried out by using the samples stained by heavy metal salt (such as phosphotungstic acid and acetic uranium) or coated by carbon. In this work, we chose the latter one to avoid the possible interference on the morphology observation caused by staining. Figure 8 shows the typical TEM

Figure 7. [η] versus Mw plots of the t-β-glucan in 0.15 M NaCl and the linear fitting curves (dashed line).

the [η] versus Mw plots of the t-β-glucan sample in 0.15 M NaCl and the linear fitting curves. It was noted that the values of Mw increased very slightly with the increase of [η] in the range of Mw > 2 × 106. This phenomenon has also appeared in the literature due to the existence of an aggregate.34,35 The Mark−Houwink equations of the t-β-glucan sample in a 0.15 M NaCl aqueous solution (lentinan concentration < 0.1 mg/mL) in the two areas were as follows [η] = 6.46 × 102M w 0.02 (2 × 106 < M w < 2 × 107)

for aggregates

(3)

for individuals

(4)

[η] = 8.79 × 10−6M w1.31 (2 × 105 < M w < 2 × 106)

In general, the exponent α is near 0 for a sphere, between 0.2 and 0.5 for solid aggregates, between 0.6 and 0.8 for a flexible polymer chain, and larger than 1 for a rigid polymer chain or a chain with helical conformation.26,36,37 Namely, the high exponent of 1.31 indicated a very stiff chain conformation, and the very low value of 0.02 suggested a sphere-like aggregate. In our findings, the α values of 0.02 and 1.31 for t-β-glucan in aqueous solution indicted aggregates and a wormlike chain, respectively. To understand the aggregated structure of polymers, the mathematical concept of a fractal rugose object has been introduced in the aggregation field. In the general theory of fractals, the fractal dimension (df) has been defined as the dependence of the total mass on the characteristic length scale on which the fractal is examined.38,39 The df value can be calculated from the α in the Mark−Houwink equation as 3/(1 + α). The df value of the t-β-glucan sample in area I was about 2.9, suggesting a 3D structure of the aggregates, which is very consistent with the sphere-like shape obtained above. The higher values of df (1.8−3.0) have been found for reversible assembly of particles into transient gel structures or reversible network structures.40−42 The high df value of 2.9 revealed that the t-β-glucan aggregated into a microgel consisted of a 3D network structure in a 0.15 M NaCl aqueous solution. This

Figure 8. Typical TEM image of the t-β-glucan.

image of the t-β-glucan chain in a dilute aqueous solution. The dark chain-like morphology indicated the rigid chains. The branched and cross-linked patterns were also observed in the image, which were likely formed as a result of overlapping and interchain hydrogen bonding between triple helical chains.13 AFM observation can be carried out under mild conditions to avoid the chain destruction.45 Figure 9 shows the AFM images of the t-β-glucan dissolved in water with different concentrations [5 (a) and 20 μg/mL (b)]. A kind of rigid linear chain can be clearly observed in Figure 9a, and the average height of the chains was measured to be 1.19 nm, which was consistant with the triple helix reported previously13 and the calculated value on the basis of the theoretical model of the triple helical chain. According to the definition of the contour length (L), the mean L (=Mw/ML) of the individual t-β-glucan chain was estimated to be 770 nm, which was comparable to that observed in the AFM image (400−1000 nm). As the polysaccharide concentration increased, a network pattern of the t-β-glucan aggregates was observed in Figure 9b. Unlike the 672

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4. CONCLUSIONS Water-soluble t-β-glucan was isolated from Lentinus edodes via a green method established in our lab and was identified to be a β-1,3-glucan with two β-1,6-D-glucopyranoside branchings for every five β-1,3-glucopyranoside linear linkages. A kind of facile method based on triple-detector SEC technology was successfully applied for determining the chain conformation of the t-β-glucan in a 0.15 M NaCl aqueous solution and in DMSO. On the basis of the Mw, Mn, ⟨S2⟩z1/2, and [η] values of the t-polysaccharide in dilute solution, the ⟨S2⟩z1/2 versus Mw and Mark−Houwink equations for the triple helix and its aggregates of t-β-glucan in a 0.15 M NaCl aqueous solution were established. The results of the molecular parameters confirmed that the t-β-glucan majorly existed as a triple helical conformation in the aqueous solution with high chain stiffness, and a few aggregates (4%) coexisted here. The fractal dimension result revealed that the aggregates existed as a reversible 3D network structure in the t-β-glucan solution. The shape of the individuals and aggregates of the t-β-glucan was supported by the results of TEM and AFM. This work provided a simple and reliable method, instead of a traditional complex and time-consuming method, for characterizing the triple helical conformation and its aggregates of complex polysaccharides.

Figure 9. AFM images of the t-β-glucan with different concentrations, (a) 5 and (b) 20 μg/mL).

side-by-side aggregation of κ-carrageenan helices during the gelation process46 or the aggregation by mismatched pairing of xanthan chains at renaturation,47 the t-β-glucan tended to form a 3D network microgel with cross-linking and overlap of the triple helical chains. The results of TEM and AFM further confirmed the existence of the reversible microgels with a 3D network structure in the t-β-glucan aqueous solution, supporting the conclusion from the df values and the conformation parameters. 3.3. Conformation Transition of β-Glucan. The conformation of the t-β-glucan in DMSO was also characterized by a SEC−MALS−RI combination. The capillary viscosity detector was not suitable for the DMSO solvent; therefore, the [η] data of the β-glucan in DMSO were not detected. Figure 10 shows the ⟨S2⟩z1/2 versus Mw plots and the

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ASSOCIATED CONTENT

S Supporting Information *

FT-IR spectrum, 13C NMR spectrum, and HMQC spectrum of t-β-glucan. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 86-27-87219274. Fax: 86-27-68754067. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, 2010CB732203), the Major Program of National Natural Science Foundation of China (21334005), and the National Natural Science Foundation of China (30530850 and 20874079).

Figure 10. ⟨S2⟩1/2 versus Mw plots for the β-glucan in DMSO and the linear fitting curve (dashed line).

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linear fitting curves of the t-β-glucan in DMSO. The exponent was calculated to be 0.48, and the relationship was given to be ⟨S2⟩z1/2 = 3.8 × 10−2Mw0.48, revealing that t-β-glucan existed as single coils in DMSO. Moreover, the average Mw value was measured to be 3.31 × 105, just one-third of Mw in the 0.15 M NaCl solution. The result further confirmed that the t-β-glucan was a triple-stranded chain in the aqueous solution and was destructed into a single random coil in DMSO. The chain conformations of the t-β-glucan in water and in DMSO were in good agreement with those in our previous works,10,11 suggesting that the present method is reliable. It was noteworthy that the characterization method was facile and fast, and the whole time required was only 6 days, about 1/30 of the time required in tradition methods. More importantly, this method can clearly reveal the structure of the aggregate.

REFERENCES

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dx.doi.org/10.1021/jp4087199 | J. Phys. Chem. B 2014, 118, 668−675

The Journal of Physical Chemistry B

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(47) Matsuda, Y.; Biyajima, Y.; Sato, T. Thermal Denaturation, Renaturation, and Aggregation of a Double-Helical Polysaccharide Xanthan in Aqueous Solution. Polym. J. 2009, 41, 526−532.

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dx.doi.org/10.1021/jp4087199 | J. Phys. Chem. B 2014, 118, 668−675