Thermally Induced Conformation Transition of Triple-Helical Lentinan

are key regulators of innate immunity responding to invading microorganisms. .... self-beating mode can be related to the normalized field-field a...
0 downloads 0 Views 1MB Size
Download PDF
J. Phys. Chem. B 2008, 112, 10343–10351

10343

Thermally Induced Conformation Transition of Triple-Helical Lentinan in NaCl Aqueous Solution Xiaohua Wang, Xiaojuan Xu, and Lina Zhang* Department of Chemistry, Wuhan UniVersity, Wuhan 430072, China ReceiVed: March 12, 2008; ReVised Manuscript ReceiVed: May 30, 2008

Lentinan, a β-(1 f 3)-D-glucan, was isolated from Lentinus edodes by using an improved extraction and purification method to show good water solubility and high yield. The results from 13C NMR, size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), and optical rotation revealed that lentinan existed in a triple-helical conformation in the aqueous solution at 25 °C, whereas the thermally induced conformation transition from triple helix to single flexible chains occurred at elevated temperatures. The dependences of the weight-average molecular weight (Mw), radius of gyration (〈s2〉z1/2), hydrodynamic radius (Rh), intrinsic viscosity ([η]), and specific optical rotation of lentinan on temperature in 0.9% NaCl aqueous solution showed an abrupt drop at 130-145 °C. It was confirmed that the conformation transitions from triple strand to single chain and from extended chains to winding chains for lentinan were completed rapidly at 130-145 °C, as a result of the simultaneous destruction of the intra- and intermolecular hydrogen bonds in lentinan. The thermally induced conformational transition was irreversible. The results from atomic force microscopy (AFM) and DLS demonstrated the existence of intrachain entanglement for the triple-helical chains, leading to the wormlike linear, circular, and crossover species for lentinan having high Mw (1.71 × 106) in aqueous solution at 25 °C. Introduction Recent findings have suggested that the polysaccharides are recognized by Toll-like receptors which are key regulators of innate immunity responding to invading microorganisms.1,2 It is believed that the immunostimulatory effect is related to the chain conformation of polysaccharides, which is usually driven by noncovalent interactions, such as van der Waals, hydrogen bonds, and hydrophobic and electrostatic forces.3,4 Schizophyllan and lentinan can activate natural immunity by promoting secretion of interleukins. It has been reported that schizophyllan having the main chain of β-(1 f 3)-D-glucan with β-(1 f 6)-Dglucosyl side chains adopts a triple-helical conformation in water and a single random coil in dimethyl sulfoxide (DMSO).5,6 Helix formation is implicated in playing an important role in protein folding, which remains one of the most challenging and difficult problems in biological science.7 Computer simulation algorithms have been developed to study the helical conformation.7,8 It is worth noting that the β-(1 f 3)-glucans can interact with certain polynucleotide to form a new triple-stranded structure, in which the helical macromolecular complexes consist of two polysaccharide strands and one polynucleotide strand.9 Schizophyllanpolynucleotide complexes have been studied, and they were characterized by a combination of hydrogen-bonding and hydrophobic interactions to form the higher-order structure.10 Moreover, ternary complexes consisting of DNA, polycation, and schizophyllan have shown high uptake efficiency when the complexes were exposed to macrophage-like cells.11 The immunopharmacological activities of schizophyllan in mice have exhibited a conformation dependence.12 The creation of new helical macromolecular complex consisting of DNA and polysaccharide would be an exciting development. Therefore, an understanding of the conformational state and its transition for * To whom correspondence should be addressed. E-mail: lnzhang@ public.wh.hb.cn. Tel: +86-27-87219274. Fax: +86-27-68754067.

the polysaccharides having helical conformation in aqueous solution is essential for their successful development and application in the fields of natural medicine and gene technology. In our laboratory, lentinan isolated from Lentinus edodes having the similar chemical structure as that of schizophyllan has been confirmed to exist in a triple helix conformation in aqueous NaCl solution, and as a single flexible chain in DMSO.13–16 We have also found that lentinan exists, respectively, as triple helices at NaOH concentration below 0.05 M and as single random-coil chains at NaOH concentration above 0.08M, at 25 °C.17 Interestingly, helical lentinan in NaCl aqueous solution exhibits relatively high inhibition ratio against the growth of Sarcoma 180 solid tumor in vivo and in vitro, whereas the bioactivities of its single flexible chains almost disappears.18 As we have known, thermal energy is another powerful tool to breaking hydrogen bonds.19–23 The thermally induced conformation transition of schizophyllan in aqueous alkaline solutions and the mixture of water and DMSO has been investigated by using high-sensitivity differential scanning calorimetry (DSC), demonstrating that the order-disorder transition temperature is dependent on the molecular weight of the biopolymer, pH, and DMSO concentration.24,25 More recently, we have successfully isolated lentinan, a β-(1 f 3)-D glucan having triple-helical structure, by using an improved extraction and purification method, showing high yield and stability for long storage time. As mentioned above, the helical structure of polysaccharides is related to their bioactivities as well as the interaction between polysaccharides and DNA. The effect of temperature on the helical structure of polysaccharides is very important for their fundamental research and clinical application in the medical and biological fields. Though the effects of changing NaOH concentration and the addition of DMSO on the chain conformation of lentinan have been investigated, the chain conformation of lentinan in water at high temperature has never been reported, nor was there any

10.1021/jp802174v CCC: $40.75  2008 American Chemical Society Published on Web 07/29/2008

10344 J. Phys. Chem. B, Vol. 112, No. 33, 2008 experimental result on effects of changing temperature on the conformational states of lentinan. In the present work, we attempted to study the thermal induced conformation transition of lentinan in 0.9% NaCl aqueous solution with static and dynamic light scattering, size exclusion chromatography combined with light scattering, viscometry, and optical rotation. Moreover, atomic force microscopy (AFM) was used to further confirm the molecular size and shape. The aim of this work is to provide meaningful information on the triple-helical structure state of lentinan in aqueous solution at different temperatures as well as the destruction of intra- and intermolecular hydrogen bonding at high temperature. Experimental Methods Sample Preparation. Lentinan was isolated from fruiting bodies of Lentinus edodes cultivated in Fujian of China by extraction with 1.25 M NaOH/0.05% NaBH4 two times, and then precipitated with 1 M acetic acid to remove R-(1 f 3)-Dglucan. The supernatant was subjected to the Sevag method to remove proteins and treated with 30% H2O2 to decolorize. It was then exhaustively dialyzed by using regenerated cellulose tube (Mw cutoff 8000) against flowing tap water for 7 days and distilled water for 3 days, and finally concentrated by rotary evaporator at reduced pressure below 44 °C. To get pure lentinan with a relatively narrow molecular weight distribution, the original solution was centrifuged at 10 000 rpm for 0.5 h, and then acetone was added dropwise into the solution to get the cloudy mixture of acetone and water (1:1 by volume) at room temperature. The resulting mixture was warmed up to 50 °C and immediately centrifuged at 10 000 rpm for 0.5 h to separate into a clear supernatant and a gel phase. The gel component was dissolved in water again to obtain a clear solution. The solution was then dialyzed against distilled water for 3 days, filtered, and finally lyophilized to give colorless flakes. The yield of the lentinan was up to about 5%; the product can be stored for a long time (more than 2 years) and it was easily soluble in water. NaCl (analytical grade) was dissolved in distilled water to prepare a 0.9% NaCl aqueous solution. DMSO (analytical grade) was treated with a molecular sieve to further dehydrate. Relatively concentrated stock solution was carefully prepared by completely dissolving the proper amount of lentinan in solvent for over 24 h with stirring. To investigate the thermally induced denaturation process at temperature higher than 80 °C, the solutions were vacuum sealed in the 10 mL glass tubes and first heated in the thermostated oil bath at the desired temperature for 20 min. After being quenched to 25 °C in an ice bath and kept for 12 h, the solutions were then used for all measurements. Every solution was filtered with 0.45 µm pore size filter (NYL, 13 mm syringe filter, Whatman, Inc., USA) before the measurements. Laser Light Scattering, SEC, and Viscometry Measurements. The scattering light intensity of lentinan in DMSO was measured by multiangle laser light scattering instrument (MALLS) equipped with a He-Ne laser (λ ) 633 nm) (DAWNDSP, Wyatt Technology Co., USA) at the angles of 49°, 56°, 63°, 71°, 81°, 90°, 99°, 109°, 118°, and 127°. The angular and concentration dependences of the scattered intensities were analyzed by using Berry’s square root plot.26

(Kc ⁄ Rθ)1 ⁄ 2 ) (1 ⁄ Mw1 ⁄ 2){1 + (1 ⁄ 6)〈s2 〉 q2 + MwA2c} (1) where K is the optical constant, c is the polymer mass concentration, q is the scattering vector, and Rθ is the reduced

Wang et al. scattering intensity at scattering angle θ. The specific refractive index increments (dn/dc) of lentinan in DMSO was measured by using an Optilab refractometer (DAWN DSP, Wyatt Technology) at 633 nm and 25 °C to be 0.058 mL/g. The Astra software (Version 4.70.07) was utilized for data acquisition and analysis. The weight-average molecular weight (Mw) and radius of gyration (〈s2〉z1/2) of lentinan in 0.9% NaCl were determined by using size-exclusion chromatography with multiangle laser light scattering (SEC-MALLS). SEC-MALLS measurements were carried out on a multiangle laser photometer mentioned above at angles of 35°, 43°, 52°, 60°, 69°, 80°, 90°, 100°, 111°, 121°, 132°, 142°, and 152° with a P100 pump (Thermo Separation Products, San Jose, CA) equipped with columns of TSK-GEL G4000 PWXL and G6000 PWXL column (7.8 mm × 300 mm, TOSOH Corp.) in 0.9% aqueous NaCl at 25 °C. A differential refractive index detector (RI-150, Thermo Separation Products, Thermo Finnigan, USA) was simultaneously connected. The eluent was 0.9% aqueous NaCl at a flow rate of 0.50 mL/min. All of the polysaccharide solutions and solvent were purified by a 0.45 µm filter and then degassed before use. The injection volume was 200 µL with a concentration of 1 mg/mL for the sample. The Astra software was utilized for data acquisition and analysis. Dynamic light scattering (DLS) was used to characterize the hydrodynamic radii (Rh) of lentinan in 0.9% NaCl aqueous solution at 25 °C. The solution was prepared at different temperatures. A modified commercial light scattering spectrometer (ALV/SP-125, ALV, Germany) equipped with an ALV5000/E multi-τ digital time correlator and a He-Ne laser (at λ ) 632.8 nm) was used at scattering angles θ ) 90°. All of the test solutions were prepared at a concentration of 1.0 × 10-4 g/mL and made optically clean by filtration through 0.45 µm Millipore filters. The precisely measured intensity-intensity time correlation function G(2)(q,τ) in the self-beating mode can be related to the normalized field-field autocorrelation function g(1)(q,τ) via the Siegert relation as27

G(2)(q, τ) ) A[1 + β|g(1)(q, τ)|2]

(2)

where A is the measured baseline and β is a constant related to the coherence of the detected optics. For a polydisperse system, g(1)(q,τ) is related to the distribution of the characteristic line width G(Γ) by27,28

|g 1 (q, τ)| ) ( )

∫0a G(Γ)e-ΓdΓ

(3)

Thus, g(1)(q,τ) can be converted to a line-width distribution G(Γ) by the CONTIN Laplace inversion algorithm in the correlator according to eq 2. For a pure diffusive relaxation, Γ is related to the translational diffusion coefficient (D), and G(Γ) can be converted to a translation diffusion coefficient distribution G(D) by

Γ ) Dq2

(4)

or a hydrodynamic radius distribution f(Rh) using the StokesEinstein equation

Rh )

kBT 6πη0D

(5)

where kB is Boltzmann’s constant, T is the temperature in K, and η0 is the solvent viscosity. The intrinsic viscosity ([η]) of lentinan solutions in 0.9% NaCl and in dry DMSO was measured at different temperatures by

Triple-Helical Lentinan in NaCl Aqueous Solution

J. Phys. Chem. B, Vol. 112, No. 33, 2008 10345

using an Ubbelohde capillary viscometer. All of the solutions had the same original concentration of 1 × 10-3 g/mL. The kinetic energy correlation was always found to be negligible. The Huggins and Kraemer equations were used to estimate the value of [η].

ηsp /c ) [η] + k[η]2c

(6)

ln ηr /c ) [η] - β[η]2c

(7)

where ηsp /c is the reduced viscosity, (ln ηr)/c is the inherent viscosity, c is the polymer concentration, and k and β are constants for a given polymer in the desired conditions. At temperatures higher than 80 °C, the solution was prepared through the above-mentioned procedures and then the measurements were made at 25 °C. Microscopy. 13C NMR spectrum of the lentinan was recorded on a Mercury 600 NMR spectrometer (Varian Inc., Palo Alto, CA) at 25 °C with TMS as the internal standard. The sample was dissolved in a mixture solution of D2O with 30 wt % DMSO-d6, as well as in pure DMSO- d6, respectively, to obtain a concentration of 10 mg/mL. The order-disorder transitions for the polysaccharides having helical structure are often accompanied by changes which may be monitored by optical rotation.29 The measurement of specific optical rotation at wavelength of 589 nm [R]589 was carried out on a Perkin-Elmer 341 polarimeter in a jacketed standard cell (10 cm/6.2 mL, Perkin-Elmer) for the lentinan solution with concentration of 1 × 10-3 g/mL. Lentinan was dissolved in 0.9% aqueous NaCl and then heated at different temperatures to obtain the desired solution for optical rotation measurements. All of the solutions were kept at 25 °C during the measurements. To provide straightforward evidence for the shape and transition of triple helix lentinan, AFM measurement was used. Lentinan was dissolved at approximately 1 mg/mL in deionized water with vigorous stirring for 24 h. All polysaccharide solutions (unheated and heated at 137 °C) were prepared through the above-mentioned procedures, and then measurements were made at 25 °C. The solutions were filtered through a 0.45 µm filter (NYL, 13 mm syringe filter, Whatman Inc., USA) and diluted with deionized water to the polymer concentration of 5 µg/mL. A 10 µL drop was deposited onto freshly cleaved mica and allowed to dry in air for 1.5 h at room temperature in a small covered Petri dish prior to imaging with the magnetically AC (MAC) mode AFM. The specimen was examined using a Picoscan atomic force microscopy (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. A piezoelectric scanner with a range of 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 humidity. The obtained image was stored as 256 × 256 point arrays.

TABLE 1: Experimental Results of Mw, of 〈s2〉z1/2, [η], and k′ for Lentian in 0.9% NaCl Aqueous Solution and in DMSO at 25°C sample

Mw × 10-4

lentinan/0.9% NaCl lentinan/DMSO

171.0 58.7

[η] 〈s2〉z1/2 (nm) (mL/g) 123.2 52.1

827.2 138.6

k′

Mw,NaCL / Mw,DMSO

0.49 0.32

2.9

NaCl aqueous solution to that in DMSO is approximately 3, which suggests triple strand chains in the aqueous solution at 25 °C. Moreover, SEC-MALLS chromatogram can produce the function of 〈s2〉z1/2 ) f(Mw). The power law of the former can be estimated from many experimental points in the SEC chromatogram. The log-log plots of 〈s2〉z1/2 against Mw for the lentinan solution unheated and preheated at 145 °C are shown in Figure 1. The two straight lines fitting the experimental points from SEC chromatogram are represented by

〈s2 〉z1⁄ 2 ) 2.04 × 10-3Mw0.75 (nm, for lentinan solution) 〈s2 〉z1⁄ 2 ) 2.41 × 10-2Mw0.55

(8)

(nm, for preheated lentinan solution) (9)

Usually, the exponent R is 0.5-0.6 for flexible chains and more than 0.6 for stiff or wormlike polymers, respectively, in a good solvent. The R values of 0.75 and 0.55 suggest that lentinan exists as a relatively stiff chain in 0.9% NaCl aqueous solution at 25 °C and as a flexible chain at 145 °C. The results mentioned above have confirmed that lentinan exists as a triple

Results and Discussion Triple Helix Conformation of Lentinan. SEC combined with LLS is convenient for the determination of Mw, 〈s2〉z1/2, and the polydispersity Mw/Mn of the polymers in solution. The results of [η], Mw, and 〈s2〉z1/2 for lentinan in 0.9% NaCl aqueous solution and in DMSO, respectively are summarized in Table 1. Clearly, the value of [η] in 0.9% NaCl aqueous solution is much higher than that in DMSO, indicating that the lentinan chains exist in completely different conformation in the two solvents. Furthermore, the ratio of Mw for the lentinan in 0.9%

Figure 1. Dependences of 〈s2〉z1/2 on Mw for lentinan in 0.9% NaCl aqueous solution at 25 °C: (a) unheated lentinan solution; (b) lentinan solution preheated to 145 °C.

10346 J. Phys. Chem. B, Vol. 112, No. 33, 2008

Wang et al.

Figure 3. 13C NMR spectra for lentinan in water/DMSO (70:30) mixtures (a) and in DMSO (b).

TABLE 2: 13C NMR Chemical Shifts of Lentinan in the Solution State chemical shift (ppm)

Figure 2. Present data of 〈s2〉z1/2 (b) and [η] (O) for lentinan in 0.9% aqueous NaCl at 25 °C, compared with the theoretical curves for the wormlike cylinder with ML ) 2180 nm-1 and q ) 100 nm (the dashed line).

helix conformation in 0.9% aqueous NaCl at 25 °C, consistent with our previous studies.14,15 Figure 2 shows theoretical curves for triple helix calculated from Yamakawa-Yoshizaki theory for a wormlike cylinder30 with molar mass per unit contour length (ML) of 2180 nm-1 and persistence length (q) of 100 nm. The dashed line and filled and open circles represent the theoretical 〈s2〉z1/2 and [η] curves as well as the present data for lentinan in 0.9% aqueous NaCl at 25 °C, respectively. Interestingly, the experimental points of the lentinan sample in NaCl aqueous solution have deviated from the theory curves for the triple helix. Our experimental results of 〈s2〉z1/2 and [η] are slightly lower than the theory data; this suggests a decrease of the chain rigidity. In our previous work,17 lentinan existed in a random coil conformation in aqueous NaOH with concentration higher than 0.08 M, and the denatured lentinan could be renatured to triple-helical conformation by dialyzing against water. Therefore, the lentinan sample in this study should be a renaturedtriplehelix,becauseitunderwentadenaturation-renaturation history during the extraction and isolation process. It has been reported that the triple-helical schizophyllan can be dissociated into random-coil chains and then subsequently renatured as triple strands in linear, circular, and branched forms by exhaustive dialysis against pure water or annealing at high temperature (but below the transition temperature).24,31,32 Thus, the decrease of the chain rigidity may result from the existence of circular and branched species in the renatured lentinan. Figure 3 shows the 13C NMR spectra for lentinan in D2O/ DMSO (70:30) mixtures and in DMSO, respectively. The

solvent

C1,C1′

C2

C3

C3′

C4

C5

C6

C6s

DMSO-d6 D2O/DMSO-d6 (70:30)

103.6 104.5

73.9 86.9 77.5 68.9 76.8 61.0 70.6 74.9 Noa 77.1 70.8 76.2 62.6 71.3

chemical shifts for the lentinan solution are summarized in Table 2. In our laboratory, 13C NMR has confirmed that lentinan exists as triple helices in D2O/DMSO (70:30), and as single flexible chains in DMSO.15 The result in Figure 3 is the same as that in ref 15. The C3 signal at 86.9 ppm for lentinan in D2O/DMSO (70:30) has disappeared, and the relative intensities of C6 signal at 61.0 ppm significantly decrease, compared with the chemical shifts for lentinan in DMSO. Moreover, the relative intensities of C3′ signal of the side chain at 77.1-77.5 ppm and substituted C6s of signal at 70.6-71.3 ppm for lentinan in D2O/DMSO (70:30) have increased. This indicates that the immobilization of the main chain by binding with intra- and intermolecular hydrogen bonds has resulted in loss of the signals of β-(1 f 3)D-linked backbone and in enhancement of the signals of the side chain. The 13C NMR results further supports the conclusion that lentinan exists as triple-stranded helical chains in aqueous solutions (including H2O/DMSO) at 25 °C. Thermally Induced Conformation Transition. It is believed that the major driving force for the helix structure of lentinan in water is due to the intramolecular hydrogen bonding, whereas that for triple-strand formation is a result of intermolecular hydrogen bonding. Usually, the values of [η] reflect the expanded extent of polymer chains. Figure 4 shows the dependence of the intrinsic viscosity [η] for lentinan in 0.9% NaCl aqueous solution on temperature (T). Clearly, the values of [η] undergo a sharp decrease in a narrow temperature range. The [η] values of lentinan at temperature bellow 130 °C are much larger than that at 145 °C and close to that at 25 °C. This suggests that the triple-helical conformation of lentinan remains intact when the temperature is lower than 130 °C. In Figure 4, [η] changes slowly at temperature higher than 145 °C and almost levels off in the range from 145 to 160 °C. Interestingly, the value of [η] for lentinan in 0.9% NaCl aqueous at 145 °C is close to that in pure DMSO at 25 °C. This suggests that lentinan changes from triple-helical conformation to single flexible chains.15 The results indicate that the intra- and intermolecular hydrogen bonds, which sustain the triple-helical structure, have been disrupted with increasing temperature, leading to the decrease of the stiffness of the chains at 145 °C in aqueous solution. Therefore, the thermally induced conformation transi-

Triple-Helical Lentinan in NaCl Aqueous Solution

Figure 4. Dependence of the intrinsic viscosity [η] for lentinan in 0.9% NaCl aqueous solution (b and O) and in DMSO (4), respectively, on temperature. Open circles are values measured directly at the corresponding temperature. Solid circles represent the [η] values measured at 25 °C for the sample after it was heated to the corresponding temperature and then suddenly cooled to 25 °C.

tion from triple helix to single flexible chain occurred, and the transition temperature (Ttran) lies in the narrow range from 130 to 145 °C. The sharp decrease of [η] in the narrow temperature range for lentinan in 0.9% NaCl aqueous solution suggests that the triple helix dissociated directly into single flexible chains during the transition process. Yanaki et al.33 have reported the dependence of [η] on the NaOH concentration (wNaOH) for a scleroglucan sample F-1 having triple helix structure in aqueous NaOH solution at 25 °C. Their value of [η] is constant up to wNaOH of 0.05 M and then decreases sharply at wNaOH of 0.1 M and levels off at a constant value at wNaOH higher than 0.2 M, indicating the dissociation of higher aggregates of the triplehelical scleroglucan at wNaOH of 0.05 M and then the breaking of trimers into single random-coil chains at wNaOH of 0.1 M. However, the [η] curve for lentinan in Figure 4 exhibits only one step at 130-145 °C, which represents the transition of triple helices to single random-coil chains. It is believed that there are no aggregates in the lentinan dilute solution at 25 °C, and the triple helix dissociated completely into single flexible chain at 145 °C in this system. As mentioned in the Experimental Methods section, the viscosity measurements for the lentinan solution heating at higher than 80 °C were carried out after quenching to 25 °C. Therefore, the helix-coil conformation transition is irreversible in this case, as shown in Figure 4. SEC chromatograms for lentinan in 0.9% NaCl aqueous solution unheated and preheated at 136 and 145 °C detected by both LLS and differential refractometer are shown in Figure 5. It is noted that the SEC chromatogram of the unheated lentinan at 25 °C contained only one peak, suggesting the existence of only one kind of chain conformation, namely the triple-helical chains. Moreover, one peak appeared in the SEC chromatogram of lentinan in aqueous solution preheated at 145 °C, whereas two peaks were observed for the sample preheated at 136 °C, corresponding to the triple-helical chains and dissociated single chains, respectively. This indicates that by raising the temperature the triple-helical lentinan changes directly to single chains in an all-or-none fashion. That is, only intact triple helices and disordered single chains coexist in the lentinan solution at about 136 °C. Destruction of Hydrogen Bonds at High Temperature. The dependences of Mw and 〈s2〉z1/2 for lentinan on temperature in aqueous solution at 25 °C are shown in Figure 6. The data of

J. Phys. Chem. B, Vol. 112, No. 33, 2008 10347

Figure 5. SEC chromatograms of lentinan in 0.9% NaCl aqueous solution (unheated, b; preheated to 136 °C, 4; preheated to 145 °C, O) at 25 °C using G6000 PWXL and G4000 PWXL columns detected by SEC-MALLS. “Detector: AUX1” represents a relative concentration.

Figure 6. Dependences of Mw (a) and 〈s2〉z1/2 (b) of Lentinan on temperature in 0.9% NaCl aqueous solution at 25 °C. Open triangle is the value measured by LLS for lentinan in DMSO at 25 °C. Solid curves are from eqs 11 and 12 with the f values estimated from the [η] data in Figure 2.

Mw and 〈s2〉z1/2 of lentinan at temperature lower than 130 °C are consistent with that at 25 °C, and the leveling-off values of Mw and 〈s2〉z1/2 at temperature higher than 145 °C are comparable to those of lentinan in pure DMSO. Clearly, the abrupt change in both Mw and 〈s2〉z1/2 for lentinan in the aqueous solution at 130 °C is attributed to the dissociation of the triple helix to

10348 J. Phys. Chem. B, Vol. 112, No. 33, 2008

Figure 7. Dependence of [R]589 of lentinan on temperature in 0.9% NaCl aqueous solution at 25 °C. Solid curve is calculated from eq 13.

single flexible chains. The Mw value of lentinan drops by a factor ∼3 on passing from 130 to 145 °C. This indicates that the lentinan molecular structure changes from triple strand to single one, as a result of the destruction of intermolecular hydrogen bonds, which sustain the trimolecular structure. The 〈s2〉z1/2 value roughly reflects the space occupied by the chains. The 〈s2〉z1/2 and [η] values at temperature lower than 130 °C are approximately 2.5 and 5.5 times as large as that at temperature higher than 145 °C. This suggests the transition of lentinan from stiff chains to flexible coil, because of the breaking of the intramolecular hydrogen bonds, which sustain the helix structure. Figure 7 shows the dependence of [R]589 on temperature for lentinan in 0.9% NaCl aqueous solution. Interestingly, with an increase of temperature, a dramatic change in the optical rotations for lentinan occurred in the temperature range from 130 to 145 °C. The change in the optical rotation was not caused by degraded lentinan, because the degraded derivatives of low molecular weight from lentinan showed the same [R]D as lentinan.34 The results from [R]D further confirm that the lentian in 0.9% NaCl aqueous solution undergoes an order-disorder conformation change at 130-145 °C, and the aggregation formed from helical chains has not occurred in the lentinan dilute solution. In view of the above results, the rapid changes of the molecular mass, size, and shape of lentinan confirm that the destruction of intra- and intermolecular hydrogen bonds have occurred simultaneously at 130-145 °C, leading to transitions from triple strands to single chains and from extended chains to winding. As mentioned in the Experimental Methods section, the denatured lentinan underwent the cycle of heating and cooling. That is, the lentinan was heated at a desired temperature and was kept there for a sufficiently long duration, exposing the polysaccharide to the condition disrupting the triple-helical structure, and was then used in all the measurements after being quenched to 25 °C. To investigate the influence to the denatured lentinan by thermal history, whether lentinan with single chains can assemble themselves, it should be taken into account in the cooling process. To clarify this point, the changes in Rh and [η] of the single flexible lentinan were investigated. Figure 8 shows the dependences of Rh and [η] on the storage time (t) at 25 °C for lentinan in 0.9% NaCl aqueous solution preheated at 145 °C for 20 min. For the polymer concentration used (c ) 1.009 × 10-3 g/mL), the values of Rh and [η] for single flexible lentinan changed hardly at the beginning, and then increased with time, indicating that no reassembly of the single flexible

Wang et al.

Figure 8. Storage time dependence of the intrinsic viscosity [η] (O) and the hydrodynamic radius Rh (b) at 25 °C for lentinan in 0.9% NaCl aqueous solution preheated at 145 °C for 20 min.

lentinan occurred at the cooling state during the measurement (within 12 h). The aggregation or self-assembly of the single flexible lentinan chains slowly occurred later. Furthermore, the SEC chromatogram for lentinan preheated at 145 °C in Figure 5 exhibited one peak. This indicates that lentinan was dissolved as single flexible chains in 0.9% NaCl aqueous solution at 145 °C, and no reassembly of the triple-helical lentinan occurred on cooling in short duration. However, for longer storage time, the values of Rh and [η] increased slowly with time. It has been discovered recently that the denatured triple-helical polysaccharide can be renatured as triple strands in the linear state, circular state, imperfections in the triplex structure or microgel clusters with the different renatured conditions.24,31,35,36 The triple-helical structure of scleroglucan was disrupted to random coil by alkaline and renatured by dialysis in short time to form a reassembled partial triple-helical structure. Then the imperfections were rearranged to linear states during subsequent longterm storage time (18 months).36 Therefore, the renatured process is very slow and depends strongly on the polymer concentration and the experimental conditions. Comparison between Experimental Results and Calculation for Chain Conformation. The data mentioned above have indicated that the dissociation of the lentinan trimers proceeded directly to single flexible chains at high temperature. If only intact triple helices (species 1) and disordered single chains (species 2) coexist in the solution at a given T, the measured [η], Mw, 〈s2〉z1/2, and [R]589 ()[R]) may be expressed by22

[n] ) f [η]1 + (1 - f )[η]2

(10)

Mw ) fMw,1 + (1 - f )Mw,2

(11)

〈s 〉zMw ) f〈s 〉z,1Mw,1 + (1 - f )〈s 〉z,2Mw,2

(12)

[R] ) f [R]1 + (1 - f )[R]2

(13)

2

2

2

Here, f denotes the weight fraction of triple helices and the subscripts 1 and 2 denote the species 1 and 2, respectively. The values of f for the lentinan in aqueous NaCl solution at different temperatures can be evaluated as a function of T from [η] in Figure 4 using eq 10 with [η]1 and [η]2 taken as the [η] values at 25 and 145 °C, respectively. The curves in Figures 6a,b and 7 show the respective properties calculated from eqs 11, 12, and 13 with those f values and the properly chosen Mw,1, Mw,2, 〈s2〉z,1, 〈s2〉z,2, and [R]1, [R]2. The satisfactory fits to the data points substantiated the validity of our estimation. That is, only intact triple helices and single flexible chains coexisted in the

Triple-Helical Lentinan in NaCl Aqueous Solution

J. Phys. Chem. B, Vol. 112, No. 33, 2008 10349

Figure 9. Distributions of the hydrodynamic radius Rh for lentinan in 0.9% NaCl aqueous solution (unheated, b; preheated at 137 °C, 4; preheated at 145 °C, O) at a fixed c of 1.0 × 10-4 g/mL at 25 °C, where θ ) 90°.

NaCl aqueous solution during the conformation transition process. This result further verified that the conformation transition took place in an all-or-none fashion. Figure 9 shows the distribution of Rh for lentinan in 0.9% NaCl aqueous solution under different thermal history but determined at 25 °C. The triangles and squares stand for the solutions preheated at 137 and 145 °C for 20 min, respectively. Clearly, the mean peak position shifted from 166.5 nm at 25 °C to 40.7 nm at 145 °C, suggesting that a chain conformation transition occurred at an elevated temperature. The Rh values were determined to be 130.8, 51.7, and 28.5 nm at 25, 137, and 145 °C, respectively. According to eq 10, the weight fraction of the triple-helical chains f was calculated to be 0.294 at 137 °C, indicating that most triple helices have been broken into single chains. Therefore, the Rh value at 137 °C can also be calculated to be 51.7 nm from eq 14.

Mw /Rh ) fMw,1 ⁄Rh,1 + (1 - f )Mw,2/Rh,2

(14)

The calculated value is in good agreement with the experimental result, giving strong evidence that triple-helical species and single chains coexist, and there is no intermediate state during the conformation transition process. This further confirms that the stiff triple-helical structure changes directly into single flexible chains at high temperature. Macromolecular Shape. It is well-known that the radius of gyration (〈s2〉z1/2) is defined as the mean square of the distance between the segment and the mass center, whereas the hydrodynamic radius (Rh) is regarded as the parameter to characterize the dimension of macromolecules in solution taking into account the hydrodynamic interactions. The molecular shape of polymers can be described from the value of 〈s2〉z1/2/Rh, which directly reflects the chain conformation.37 Usually, 〈s2〉z1/2/Rh is about 0.77 for a uniform and nondraining sphere; 1.0-1.1 for a loosely connected hyperbranched chain or aggregate; 1.5-1.8 for a flexible chain in a good solvent; and more than 2.0 for an extended rigid chain. Interestingly, the values of 〈s2〉z1/2/Rh for lentinan in NaCl aqueous solution were calculated from the data in Figures 6 and 8 to be 0.94 at 25 °C for the unheated sample, 1.74 for the sample preheated at 137 °C, and 1.76 for the sample preheated at 145 °C. This suggests that lentinan existed as flexible chains in the NaCl aqueous solution preheated at 145 °C. At 137 °C, the value of 〈s2〉z1/2/Rh (1.74) is also indicative of a flexible chain conformation. A majority of the lentinan molecules (∼70%) exists as single chains under this condition.

On the basis of the results from SEC-MALLS and viscometry, it can be concluded that lentinan exists as a stiff triple-helical chain in 0.9% NaCl solution at 25 °C, and there is no aggregates in the dilute solution of lentinan. It is noted that the value of 〈s2〉z1/2/Rh for the unheated sample at 25 °C is ∼0.94, similar to the value of loosely connected hyperbranched chain or aggregate of normal polymers. This 〈s2〉z1/2/Rh value may be related to the contribution of the winding triple-strand chains and their intrachain entanglement. The chains of lentinan are very long because of the high molecular weight (Mw ) 1.71 × 106), leading to the winding propensities of the triple helix. Usually, the circular morphology of polysaccharides possesses smaller radius of gyration than the extended rodlike chains, resulting in the reduction of the 〈s2〉z1/2/Rh value. It is believed that the triple-helical lentinan exists as temporary intrachain entanglement and winding chain conformation in the aqueous solution at 25 °C. AFM has become an invaluable metrological tool to characterize surface topology on the nanometer scale particles, macromolecules absorbed to surfaces, biopolymer conformation, and the linear and circular triple helix structures of β-(1 f 3)-Dglucans.35,38,39 To confirm the above supposition that the triplehelical lentinan exists as winding chains caused by intrachain entanglement, AFM was used to determine the shape of the chains under different conditions. According to our previous work,14,15,40 the conformation of lentinan is not sensitive to the concentration of NaCl, i.e., the lentinan exists as a triple-helical chain in aqueous solution and also in NaCl solution. Moreover, salt will crystallize and cover the lentinan chains at the process of drying on mica, which may interfere with the AFM images of lentinan. Therefore, we used deionized water to prepare the samples for AFM. Figure 10 shows the MAC mode AFM images of the unheated and preheated lentinan samples at 137 °C in deionized water. It appears that the lentinan molecules in water at 25 °C exhibited the wormlike linear, circular, and crossover species (i.e., multichain clusters) as shown in Figure 10, a and b. The circular and cross species have been also observed at the denatured-renatured scleroglucan38,39,41,42 and schizophyllan.31,32 It has been reported that the SEC-MALLS and the HPLC chromatogram of scleroglucan following alkaline denaturation and subsequently renaturation by neutralization or thermal annealing appeared as a broad peak with a shoulder, corresponding to linear and circular states, respectively, suggesting the linear and circular molecules have different elution volumes.36,42,43 Clearly, there are substantial differences between the SEC chromatograms of lentinan in this study and scleroglucan. As shown in Figure 5, the SEC chromatogram of the unheated lentinan at 25 °C contains one peak, which represents the existence of only one kind of helical state. Meanwhile, the results from SEC-MALLS and viscometry demonstrated that there are no aggregates in the lentinan dilute solution. By analogy, we therefore suggest that the circular and cross morphology of the lentinan can be explained by the temporary intrachain entanglement, namely self-entanglement, which occurred when a long macromolecular chain created crossover or superposition by bending the chain into itself. Therefore, the triple-helical lentinan exists as bended chains rather than as extended rigid chains in the aqueous solution at 25 °C, when it has relatively high Mw. From Figure 10a, the measured mean thickness of lentinan is ∼1.21 nm averaged over hundreds of molecules, which is approximately 65% of the triple-strand thickness of a lentinan molecule with a center-to-center spacing of 1.73 nm expected from X-ray diffraction.44 It is worth noting that some cross and circular images displayed the mean

10350 J. Phys. Chem. B, Vol. 112, No. 33, 2008

Wang et al.

Figure 10. MAC mode AFM topographic images of the lentinan solution at 25 °C (a, b) and preheated at 137 °C (c) deposited on mica as 5 µg/mL solution in pure water, respectively. The z scale is as shown. Trace across through two linear triple-helical chains shown above as horizontal white bars (bottom).

Figure 11. Schematic representation of the thermally induced conformation transition of lentinan solution at 25 °C (a), preheated at 137 °C (b), and preheated at 145 °C (c) in NaCl aqueous solution.

thickness of 1.6-2.4 nm, suggesting that the triple-helical chains have been winded to overlap or changed to double and even triple circles. The contour length (L) of the triple helix lentinan can be calculated from the molar mass per unit contour length (ML ) 2200 nm-1).14,17 According to the definition for a wormlike chain, the value of L (L ) Mw/ML) for lentinan in aqueous solution at 25 °C has been obtained to be about 770 nm, which is consistent with that observed in the AFM image. Figure 10c shows the AFM image of the lentinan preheated at 137 °C. There are relatively short linear and small circular traces, The intact triple-helical chains for the lentinan at 137 °C are much fewer than that at 25 °C (Figure 10a). This suggests that a few of the slight intrachain entanglement of the lentinan triple helices still existed in the aqueous solution at 137 °C. The results from DLS and AFM have revealed that the predominant species of the triple-helical lentinan in aqueous solution exists as wormlike linear, circular, and crossover species, and the disentanglement and transition from the triple

helix to single flexible chain occurred simultaneously at 130-145 °C. On the basis of the results from SEC-MALLS, DLS, viscometry, and AFM, a schematic description of the thermally induced transition of lentinan in the aqueous solution is proposed in Figure 11. The triple-helical chains exist as slight intrachainentangled species in the aqueous solution at room temperature as shown in Figure 11a, in which they are winded to form wormlike linear, circular and crossover shapes as detected with DLS in Figure 10a. With an increase in temperature from 25 to 137 °C, a sharp change from triple helix to disordered single chain appears, accompanying the dissociation of the chains. As shown in Figure 11b, triple-helical chains and single coil chains coexist at 130-145 °C, leading to the predominant species of the short chain and small trace as shown in Figure 10c, because strand separated segments, either internally or at the terminals, have not been clearly identified.36 As the temperature is raised to 145 °C, the triple helices dissociate completely into single

Triple-Helical Lentinan in NaCl Aqueous Solution flexible chains as shown in Figure 11c. This work, for the first time, demonstrates that the triple-helical lentinan exists as intrachain-entangled species in aqueous solution. Conclusions Water-soluble β-(1 f 3)-D-glucan (lentinan) existed as triplehelical chains in 0.9% NaCl aqueous solution at 25 °C. In view of the conformation parameters, the triple-helical chains of lentinan exhibited the winding and bended shape rather than extended rigid state in the aqueous solution. The triple-helical chains were sufficiently long to result in the intrachain entanglement via winding of the backbone. The triple helix directly dissociated to single flexible chains in an all-or-none fashion at 130-145 °C, confirming that the intra- and intermolecular hydrogen bonds of lentinan were destroyed simultaneously. The AFM images confirmed that the triple-helical chains of lentinan with high Mw formed wormlike linear, circular, and crossover species in aqueous solution at 25 °C, as a result of the slight intra-entanglement of the long chain. The triple helix-single coil chain transition of the lentinan occurred sharply in a narrow temperature from 130 to 145 °C. Furthermore, the thermally induced conformation transition was irreversible. The information about the molecular mass, size, and shape, and the intraentanglement of the triple-helical chains as well as their dissociation at elevated temperature, could be obtained from DLS, LLS, SEC-MALLS, and AFM. The thermally induced conformation transition of lentinan from triple strand to single one and from extended chain to winding will be important for the investigation of the natural immunity of polysaccharides. Acknowledgment. We gratefully acknowledge the major grant of the National Natural Science Foundation of China (30530850), the National Natural Science Foundation (20474048 and 20404010), and the High-Technology Research and Development Program of China (2006AA02Z102). The authors thank Prof. Daiwen Pang and Dr. Yi Lin, Wuhan University, for providing the AFM measurements. References and Notes (1) Takeda, K.; Akira, S. Int. Immunol. 2005, 17, 1–14. (2) Young, S. H.; Ye, J. P.; Frazer, D. G.; Shi, X. L.; Castranova, V. J. Biol. Chem. 2001, 276, 20781–20787. (3) Yin, Y.; Zhang, H.; Nishinari, K. J. Phys. Chem. B 2007, 111, 1590–1596. (4) Perico, A.; Mormino, M.; Urbani, R.; Cesaro, A.; Tylianakis, E.; Dais, P.; Brant, D. A. J. Phys. Chem. B 1999, 103, 8162–8171. (5) Norisuye, T.; Yanaki, T.; Fujita, H. J. Polym. Sci.: Polym. Phys. Ed. 1980, 18, 547–558. (6) Yanaki, T.; Norisuye, T.; Fujita, H. Macromolecules 1980, 13, 1462–1466. (7) Wu, X.; Wang, S. J. Phys. Chem. B 2001, 105, 2227–2235. (8) Mahadevan, J.; Lee, K. H.; Kuczera, K. J. Phys. Chem. B 2001, 105, 1863–1876.

J. Phys. Chem. B, Vol. 112, No. 33, 2008 10351 (9) Uezu, K.; Numata, M.; Hasegawa, T.; Li, C.; Kaneko, K.; Shinkai, S. Chem. Commun. 2005, 5, 4383–4398. (10) Sakurai, K.; Mizu, M.; Shinkai, S. Biomacromolecules 2001, 2, 641–650. (11) Takeda, Y.; Shimada, N.; Kaneko, K.; Shinkai, S.; Sakurai, K. Biomacromolecules 2007, 8, 1178–1186. (12) Ohno, N.; Miura, N. N.; Chiba, N.; Adachi, Y.; Yadomae, T. Biol. Pharm. Bull. 1995, 18, 1242–1247. (13) Zhang, P.; Zhang, L.; Cheng, S. Biosci. Biotechnol. Biochem. 1999, 63, 1197–1202. (14) Zhang, L.; Zhang, X.; Zhou, Q.; Zhang, M.; Li, X. Polym. J. 2001, 33, 317–321. (15) Zhang, L.; Li, X.; Zhou, Q.; Zhang, X.; Chen, R. Polym. J. 2002, 34, 443–449. (16) Zhang L.; Xu X.; Unursaikhan, S.;, Li, X. China Patent 200410060850.7. (17) Zhang, X.; Zhang, L.; Xu, X. Biopolymers 2004, 75, 187–195. (18) Zhang, L.; Li, X.; Xu, X.; Zeng, F. Carbohydr. Res. 2005, 340, 1515–1521. (19) Tada, T.; Matsumoto, T.; Masuda, T. Biopolymers 1997, 42, 479– 487. (20) Tada, T.; Matsumoto, T.; Masuda, T. Chem. Phys. 1998, 228, 157– 166. (21) Yanaki, T.; Tabata, K.; Kojima, T. Carbohydr. Polym. 1985, 5, 275–283. (22) Nakanishi, T.; Norisuye, T. Biomacromolecules 2003, 4, 736–742. (23) Sato, T.; Norisuye, T.; Fujita, H. Carbohydr. Res. 1981, 95, 195– 203. (24) Kitamura, S.; Hirano, T.; Takeo, K.; Fukada, H.; Takahashi, K.; Falch, B. H.; Stokke, B. T. Biopolymers 1996, 39, 407–416. (25) Kitamura, S.; Kuge, T. Biopolymers 1989, 28, 639–654. (26) Berry, G. C. J. Chem. Phys. 1966, 44, 4550–4564. (27) Berne, B.; Pecora, R. Dynamic Light Scattering; Plenum Press: New York, 1976. (28) Chu, B. Laser Light Scattering, 2nd ed.; Academic Press: New York, 1991. (29) Goodall, D. M.; Norton, I. T. Acc. Chem. Res. 1987, 20, 59–65. (30) Yamakawa, H.; Yoshizaki, T. Macromolecules 1980, 13, 633–643. (31) Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kitamura, S. Macromolecules 1991, 24, 6349–6351. (32) Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kuge, T.; Kitamura, S. Biopolymers 1993, 33, 193–198. (33) Yanaki, T.; Norisuye, T. Polym J. 1983, 15, 389–396. (34) Sasaki, T.; Takasuka, N.; Chihara, G.; Maeda, Y. Y. Gann 1976, 67, 191–195. (35) McIntire, T. M.; Brant, D. A. J. Am. Chem. Soc. 1998, 120, 6909– 6919. (36) Falch, B. H.; Stokke, B. T. Carbohydr. Polym. 2001, 44, 113–121. (37) Niu, A.; Liaw, D. J.; Sang, H. C.; Wu, C. Macromolecules 2000, 33, 3492–3494. (38) McIntire, T. M.; Penner, R. M.; Brant, D. A. Macromolecules 1995, 28, 6375–6377. (39) McIntire, T. M.; Brant, D. A. Biopolymers 1997, 42, 133–146. (40) Zhang, Y.; Xu, X.; Xu, J.; Zhang, L. Polymer 2007, 48, 6681– 6690. (41) Vuppu, A. K.; Garcia, A. A.; Vernia, C. Biopolymers 1997, 42, 89–100. (42) Falch, B. H.; Elgsaeter, A.; Stokke, B. T. Biopolymers 1999, 50, 496–512. (43) Sletmoen, M.; Christensen, B. E.; Stokke, B. T. Carbohydr. Res. 2005, 340, 971–979. (44) Bluhm, T. L.; Sarko, A. Can. J. Chem. 1977, 55, 293–299.

JP802174V