Multiple Conformation Transitions of Triple Helical Lentinan in DMSO

Jul 1, 2009 - The hydrogen bonding interactions of the triple helical lentinan, β-(1→3)-d-glucan from Lentinus edodes, in the mixtures of dimethyl ...
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J. Phys. Chem. B 2009, 113, 9915–9923

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Multiple Conformation Transitions of Triple Helical Lentinan in DMSO/Water by Microcalorimetry Xiaohua Wang,† Yangyang Zhang,† Lina Zhang,*,† and Yanwei Ding‡ Department of Chemistry, Wuhan UniVersity, Wuhan 430072, China, and Hefei National Laboratory for Physical Sciences at Microscale, Department of Chemical Physics, UniVersity of Science and Technology of China, Hefei 230026, China

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The hydrogen bonding interactions of the triple helical lentinan, β-(1f3)-D-glucan from Lentinus edodes, in the mixtures of dimethyl sulfoxide (DMSO) and water were investigated by light scattering, viscometry, and ultrasensitive differential scanning calorimetry (US-DSC). The results revealed that two conformation transitions occurred in the lentinan solution with an increase of temperature. A reversible transition from triple helix I to triple helix II, namely, from a high degree of immobilization of the backbone to a more freely rotating one, occurred in the temperature range from 8 to 45 °C. The other was an irreversible conformation transition from triple helix to single strand flexible chain at 90-140 °C, depending on the DMSO concentration. The side chain of the triple helix I lentinan combined with water clusters through hydrogen bonds to form an associating water layer in the first region, leading to a high degree of immobilization of the backbone. However, the side chain could rotate in triple helix II at slightly elevated temperature, as a result of the breaking of the associating structure with relatively lower energy. The second transition resulted from the destruction of the intra- and intermolecular hydrogen bonds in lentinan, which sustain the triple helical structure. Furthermore, the transition in the high temperature region showed high cooperation, suggesting that the intra- and intermolecular hydrogen bonds with the relatively high energy were destructed simultaneously. Therefore, the diversity of the hydrogen bonds created the multiple conformational transitions of the triple helical polysaccharide in the aqueous solution. Introduction Some fungal polysaccharides from the β-(1f3)-D-glucan family are regarded as biological response modifiers due to the immune stimulating effect,1–3 and they have shown antitumor and anti-AIDS viral activity in humans.4–6 Recent findings have revealed that the β-(1f3)-D-glucan with triple-strand helical structures such as schizophyllan can form complexes with polynucleotides, carbon nanotubes, and some hydrophobic polymers.7–9 Moreover, schizophyllan could be an excellent carrier for immunostimulatory CpG-DNAs to enhance cytokine secretion.10,11 The triple helix structure of β-(1f3)-D-glucan is important in the complexation with the polynucleotides, and the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family.4 Lentinan is a neutral polysaccharide extracted from the fruiting body of Lentinus edodes, which has a similar structure to schizophyllan. It consists of a β-(1f3)-D-linked backbone of glucose residues, with two β-(1f6)-D-glucosyl residues for every five main-chain glucose residues.12,13 The average degree of branching is about 27% calculated from the peak area ratio in 13C NMR spectra. Interestingly, helical lentinan in NaCl aqueous solution has exhibited a 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 decreased.14 It has been reported that β-glucan can bind with cell receptors through the hydrogen bonds or electrostatic * To whom correspondence should be addressed. E-mail: lnzhang@ public.wh.hb.cn. Phone: +86-27-87219274. Fax: +86-27-68754067. † Wuhan University. ‡ University of Science and Technology of China.

interaction, leading to the activation of the immune cell and the production of cytokines, so as to enhance the immune function.15,16 Moreover, there are abundant evidence that lentinan can promote human health by stimulating the immune system, and this stimulation has been used to treat various forms of cancers and AIDS.17,18 Thus, the understanding of the chain conformations of lentinan and the mechanism of the interactions with tumor cells has attracted much attention from biologists and polymer scientists.17–22 In our laboratory, lentinan has been confirmed to exist as triple helical chains in NaCl aqueous solution and as a single flexible chain in dimethyl sulfoxide (DMSO),23 and it undergoes a transition from triple helix to single coil chain when the water composition decreases to about 0.15 in mixtures of water and DMSO at 25 °C.24 Lentinan exhibits high stability over a wide range of temperature and pH but underwent rapid conformation transitions under certain conditions.25,26 The helix-coil transition of the lentinan occurred sharply in the range of the NaOH concentration (wNaOH) from 0.05 to 0.08 M. Moreover, thermally induced conformational transitions from triple helix to single coil appear at about 130-145 °C.27 It is well-known that the triple helical structure of lentinan is sustained and stabilized by the intra- and intermolecular hydrogen bonds in aqueous solution. It is noted that the denatured lentinan (single flexible chains) can be renatured to triple helical conformation by dialyzing against water for a long time,25 and its collapsed coil chains are very prone to form large aggregates,26 suggesting that strong hydrogen bonding interactions exist in lentinan. It is not hard to imagine that the enhancement of the immune function may be related to the hydrogen bonds of lentinan, which are very important to reveal the mechanism of its physiological

10.1021/jp811289y CCC: $40.75  2009 American Chemical Society Published on Web 07/01/2009

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function. Therefore, we are interested in the studies on the conformation and its change of triple helical β-(1f3)-D-glucan in the aqueous solution through the hydrogen bonds under different conditions. Ultrasensitive differential scanning calorimetry (US-DSC) is a powerful tool to study the hydrogen bond interactions, which can measure energy changes as small as 0.02 µcal/s involved in a dynamic process of a macromolecular chain in solution.28–33 However, the diversity of the hydrogen bonds of lentinan in aqueous solution studied by US-DSC has never been reported. The aim of the present work is to investigate the variation of hydrogen bonding and multiple conformation transitions of lentinan in mixtures of water and DMSO over the entire composition range by US-DSC. The interpretation of the calorimetric data is based on a phenomenological model of the transition, in which a macromolecule is viewed as consisting of independent “cooperative units” and the transition in each of these units is regarded as a temperature-induced all-or-none transition from one state (e.g., the triple helical state of the cooperative unit) to another state (e.g., the single coil state of the cooperative unit).34 Our findings may lead to a better understanding of the diversity of the hydrogen bonding interactions in the triple helical polysaccharides. Experimental Methods Sample Preparation. Lentinan was isolated from the fruiting bodies of Lentinus edodes, a commercial product cultivated in Fujian, China, by extraction with 1.25 M NaOH/ 0.05% NaBH4. The detailed procedure of extraction was reported previously.27 The resulting samples were further fractionated by repeated precipitation from water to acetone at room temperature to obtain several fractions. The precipitates were redissolved in water to obtain clear solutions, which were filtered and lyophilized for 1 week. The middle part was chosen in the present work. DMSO (analytical grade) was treated with a molecular sieve to further dehydrate. By changing the weight fraction of DMSO in the mixture (wDMSO), a series of mixed aqueous solutions with different wDMSO values were prepared. The lentinan sample was dissolved, respectively, in distilled water, DMSO, and the DMSO/water mixtures to prepare the polymer solutions. A concentrated stock solution was carefully prepared by completely dissolving the proper amount of lentinan in the appropriate solvent for over 24 h with stirring at room temperature. The preparation of polysaccharide solutions was done under the same conditions, such as stirring, dissolving, and residence time. Laser Light Scattering and SEC-LLS Measurements. The scattering light intensity of lentinan in DMSO was measured by a multiangle laser light scattering (MALLS) instrument equipped with a He-Ne laser (λ ) 633 nm) (DAWNDSP, Wyatt Technology Co., Santa Barbara, CA) at multiple angles (θ) in the range from 49 to 127° at 25 °C. The basic light scattering equation is as follows35

(

( )) + 2A c + ...

16π2〈S2〉z 2 θ Kc 1 sin ) 1+ Rθ Mw 2 3λ2

2

(1)

where K is an optical constant equal to [4π2n2(dn/dc)2]/(λ4NA); c, the polymer concentration in mg/mL; Rθ, the Rayleigh ratio; λ, the wavelength; n, the refractive index of the solvent; dn/dc, the refractive index increment; NA, Avogadro’s number; and A2, the second virial coefficient. The polysac-

charide solution with the desired concentration was prepared, and optical clarification of the solutions was achieved by filtration through a 0.45 µm pore size filter (PTFE, Puradisc 13-mm Syringe Filters, Whatman, England) into a scattering cell. The specific refractive index increments (dn/dc) of lentinan in water and DMSO were measured by using an Optilab refractometer (DAWN DSP, Wyatt Technology) at 633 nm and 25 °C to be 0.140 and 0.058 mL/g, respectively. The Astra software (version 4.90.07) was utilized for data acquisition and analysis. The weight-average molecular weight (Mw) and radius of gyration (〈s2〉z1/2) of lentinan in distilled water were determined by using size exclusion chromatography with multiangle laser light scattering (SEC-MALLS). The SEC-MALLS measurements were carried out on the multiangle laser photometer mentioned above at angles of 43, 52, 60, 69, 80, 90, 100, 111, 121, 132, and 142° combined with a P100 pump (Thermo Separation Products, San Jose, CA) equipped with columns of TSK-GEL G4000 PWXL and G6000 PWXL (7.8 mm × 300 mm, TOSOH Corporation) in distilled water at 25 °C. A differential refractive index detector (RI-150, Thermo Separation Products, Thermo Finnigan, U.S.A.) was simultaneously connected. The eluent was double distilled water with a flow rate of 0.8 mL/min. All of the polysaccharide solutions and the 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. Viscosity Measurement. The viscosities of the lentinan solutions in distilled water, dry DMSO, and the water/DMSO mixtures were measured at different temperatures by using an Ubbelohde capillary viscometer. All of the solutions had the same original concentration of 0.5 × 10-3 g/mL. The kinetic energy correlation was found to be negligible. The Huggins and Kraemer equations were used to estimate the value of the intrinsic viscosity ([η]).

ηsp /c ) [η] + k[η]2c

(2)

ln ηr /c ) [η] - β[η]2c

(3)

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 under the desired conditions. The changes of (ln ηr)/c with temperature for lentinan in the mixtures of water and DMSO with different wDMSO values were measured. At temperatures higher than 80 °C, the solution was vacuum-sealed in the 10 mL glass tubes and then heated in the thermostatted 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 solution was then used for viscosity measurements at 25 °C. Ultrasensitive Differential Scanning Calorimeter. US-DSC measurements were performed on a VP-DSC microcalorimeter (VP-DSC, Microcal Inc., U.S.A.) with solvents as the references. The concentrations of lentinan in the mixtures of DMSO and water were 0.5, 1.0, and 5.0 mg/mL. The sample solution was heated from 5 to 130 °C at a heating rate of 0.5 °C/min. Data analysis was done by using Microcal software. The excess specific heat capacity was obtained by using the progress baseline as the reference. The transition temperature (Tm) was taken as that corresponding to the maximum excess specific heat capacity during the transition. The “real” calorimetric enthalpy change (∆Hcal) during the transition was calculated

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TABLE 1: Experimental Results of Lentinan in Aqueous Solution and Pure DMSO by SEC-MALLS, LLS, and Viscometry at 25°C solvent

Mw × 10-4

〈s2〉z1/2 (nm)

[η] (mL/g)

Mw,water/Mw,DMSO

[η]water/[η]DMSO

H2 O DMSO

163.5 52.0

123.2 62.1

1014.2 130.7

3.1

7.6

by integration of the peak area. The errors for the transition temperature and enthalpy were within (0.1 and (1%, respectively. DSC data analysis was referred to the methods applied to (or used for) polymers, proteins, and polysaccharides.34,36–38 Results and Discussion The Chain Conformation Transition. Table 1 summarizes the values of [η], Mw, and 〈s2〉z1/2 for lentinan in distilled water and DMSO at 25 °C determined by light scattering and viscometry, respectively. The polydispersity (Mw/Mn) is 1.20, indicating the resultant lentinan sample has a relatively narrow molecular weight distribution. As expected, the ratio of Mw in water and in DMSO is determined to be 3.1, whereas the ratio of [η] in water and in DMSO is 7.6, confirming that the lentinan sample exists as a triple helix in water and a single random coil in pure DMSO.24,27 The value of [η] reflects the hydrodynamic volume of polymers and the extent of chain expansion. The sharp decrease of viscosity is attributed to the dissociation from triple strand to single chain, as well as the transition from extended stiff chains to flexible winding chains. The lentinan sample was dissolved in the DMSO/water mixtures with different weight fractions of DMSO (wDMSO). The polysaccharide solutions with a given concentration (c ) 3.0 × 10-4 g/mL) were prepared, and then, they were used for viscosity measurements. Figure 1 shows the dependence of (ln ηr)/c for lentinan in the DMSO/water mixtures on temperature (T). For wDMSO < 0.85, all of the (ln ηr)/c values underwent a sharp decrease at different temperatures, and the transition temperature decreased dramatically with an increase of wDMSO. Furthermore, in a narrow DMSO concentration range (wDMSO ) 0.70-0.85), the transition temperature abruptly decreased with increasing wDMSO. In our previous work,27 lentinan was confirmed to undergo an irreversible conformation transition from triple helix to single coils in aqueous solution at a temperature higher than 130 °C. Therefore, the dramatic decrease of (ln ηr)/c for lentinan in the DMSO/water mixtures suggested that the lentinan has changed from a triple helical conformation to single flexible chains. Interestingly, for wDMSO

Figure 1. Dependence of (ln ηr)/c for lentinan in DMSO/water mixtures on temperature: distilled water (O); wDMSO of 0.70 (b); wDMSO of 0.78 (4); wDMSO of 0.81 (1); wDMSO of 0.85 (0); wDMSO of 0.90 (9).

g 0.85, the (ln ηr)/c values were very low and hardly changed with temperature. The explanation is that, at wDMSO g 0.85, lentinan has already transformed into single random coils at 25 °C.24 Figure 2 shows the temperature dependence of the excess heat capacity for lentinan in DMSO/water at a polysaccharide solution concentration of 5 mg/mL. Clearly, two endothermic peaks appeared in the US-DSC curves, indicating two transitions in the chain structure of lentinan. This experimental result was similar to those of the triple helical schizophyllan in the mixtures of water and DMSO.36 The triple helical schizophyllan has been confirmed to undergo the transition from a highly ordered triple helix to a disordered one by optical rotation (ORD) and circular dichroism (CD) measurements. The transitions are caused by changes in the hydrogen bonding between water and the side chain of polysaccharide.37,38 Our results showed that the first transition peak (T1) for lentinan appeared at a temperature lower than 45 °C. We define T1 as the transition from triple helix I to triple helix II. As shown in Figure 1, the viscosity of lentinan solutions hardly changed with T in the low temperature range. It suggested that the gross morphology and size of triple helical molecules for triple helix I and II are similar, and the slight difference could not be detected by viscometry. The second transition peak of lentinan in water is consistent with the thermal transition temperature (130-145 °C) obtained in our previous work,27 indicating that lentinan changes from a triple helical conformation to single flexible chains. Thus, lentinan in DMSO/ water underwent the transition from triple helix to single random coil at temperatures higher than 80 °C (high temperature region), referred to in this work as the second transition peak (T2). Conformational Transition at Lower Temperature. When wDMSO was lower than 0.70, the transition temperature (Tm1) for lentinan in the DMSO/water in the lower temperature region was elevated with increasing wDMSO, whereas, when wDMSO was higher than 0.70, Tm1 rapidly decreased with increasing wDMSO, as shown in Figure 2. Thus, the T1 transition corresponds to the change from triple helix I to triple helix II for lentinan. Figure 3 shows the temperature dependence of the specific heat capacity (Cp) of lentinan in DMSO/water at wDMSO ) 0.7799 with a polysaccharide concentration of 5.02 × 10-3 g/mL in the lower temperature region. The difference between the heat

Figure 2. Temperature dependence of the excess heat capacity for lentinan in DMSO/water mixtures detected by VP-DSC. The wDMSO values are indicated in the figure.

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Figure 3. Temperature dependence of the specific heat capacity (Cp) of lentinan in a DMSO/water mixture with a wDMSO value of 0.7799 in the lower temperature region detected by VP-DSC.

Figure 4. 13C NMR spectra for lentinan in a DMSO/D2O mixture (wDMSO ) 0.30).27

capacities of the triple helix I and the triple helix II can be measured as the difference between Cp(T) curves extrapolated from both sides of the transition (i.e., from the triple helix I and triple helix II states) to its mid point. Interestingly, both the values of Cp(T) for lentinan before and after the transition show an almost linear decrease with the increasing temperature. The low Cp(T) value is related to the decrease of the number of polymer-water contacts.34 Usually, the solvation is enhanced with elevating temperature, favoring the extended conformation of polymer molecular chains in solution. Moreover, the chain segment motion increases with elevated temperature, leading to the greater flexibility of the molecular chain. The transition temperature corresponding to the first transition peak of lentinan in the range of 5-60 °C rises with the increasing wDMSO up to about 45 °C at wDMSO ) 0.7 and then more abruptly decreases with the continuous increase of wDMSO. We can surmise that a protective layer formed by DMSO around the triple helical chain of lentinan prevents the change from triple helix I to triple helix II. In order to determine how the conformation of lentinan changes in the first transition, we analyzed the 13C NMR spectra for lentinan in DMSO/D2O mixtures before the first transition (25 °C), as shown in Figure 4.27 The absence of the C3 signal for the lentinan in the DMSO/D2O with wDMSO ) 0.3 indicated the high immobilization of the backbone.24 It is worth noting that water molecules exist as clusters, which is easy to associate with the molecules containing the hydroxyl groups to form complexes via hydrogen bonding, and the complex is relatively stable at low temperature.39,40 It is well-known that many β-(1f3)-D-glucans extracted from fungus, especially the linear glucans, are insoluble in water. Curdlan is slightly soluble in water when the molecular weight is relatively high, whereas schizophyllan can be solvated in sufficient quantities to afford liquid crystals.4 However, the lentinan with such high molecular weight can be dissolved in water. This good solubility of polysaccharides should be induced mainly by the side chains

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Figure 5. Dependence of Tm1 for lentinan in DMSO/water mixtures on wDMSO in the low temperature region. Polysaccharide concentration: (O) c ≈ 5.0 mg/mL; (4) c ≈ 1.0 mg/mL.

(glucose residues). The water solubility of lentinan gives a strong proof that the hydroxyl of the side chain associates with water clusters, leading to good dissolution in aqueous solution. Two types of hydrogen bonding via water molecules, the side-chain/ main-chain and the side-chain/side-chain hydrogen bonding, play an important role in determination of the triple helix conformation, and the motion of water molecules around the side chains is much more hindered due to hydrogen bonding between the glycosyl side chains and the water.4 Thus, we proposed that the surrounding water clusters combine with the side chain of lentinan through hydrogen bonds to form a relatively stable network (i.e., an associating water layer) at low temperature. The phenomenon is similar to the model of the triple helical polysaccharide schizophyllan.37 From the analysis of 13C NMR spectra and the value of (ln ηr)/c in Figure 1, it can be indicated that, before the T1 transition, lentinan exists as a triple helix in solution, as a result of the relatively stable hydrogen bonding network between side-chain hydroxyl and water clusters, leading to the highly immobile backbone of triple helix I. When the temperature increases, the hydrogen bonding network is broken easily to perturb the associating layer, leading to the increase of the free rotation of the side chain and backbone of triple helix. The hydrogen bonds between the side chain of lentinan and the surrounding water clusters were destroyed, and the side chains changed from order to disorder, resulting in the decrease of the number of polymer-water contacts. Thus, the present decrease in Cp further reflects the increase of the winding of molecular chains after transition. Therefore, in the first conformation transition region, lentinan changes from the triple helix I with a highly immobilized backbone to the relatively free triple helix II with an increase of temperature from 8 to 45 °C. Figure 5 shows the dependence of the transition temperature (Tm1) at lower temperature on wDMSO for lentinan in the DMSO/ water mixtures with two different polysaccharide concentrations (c ) 1.0 and 5.0 mg/mL). Tm is the temperature at which the maximal excess specific heat capacity occurs in the transition process. The Tm value of lentinan at lower temperature is labeled as Tm1. Clearly, when wDMSO < 0.7, Tm1 increased with the increasing wDMSO; when wDMSO ) 0.7, the value of Tm1 was at the maximum; and then when wDMSO > 0.7, Tm1 rapidly decreased. The addition of DMSO has two kinds of effects on the associating water layer of lentinan. When the DMSO concentration is relatively low, it is not sufficient to destroy the association structure of triple helix-water. Due to the solvation, DMSO only surrounds the outside to protect the associating water layer, leading to the shift of Tm1 to high temperature. However, when wDMSO was higher than 0.7, DMSO

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TABLE 2: Calorimetric Parameters of the Transition at Lower Temperature for Lentinan in DMSO/Water Mixtures wDMSO 0.0 0.1998 0.3998 0.6004 0.6997 0.7799 0.8116 0.8299 0.8495

c (mg/mL)

Tm1 (°C)

Qtr (J g-1)

∆Hcal (kJ mol-1)

∆HvH (kJ mol-1)

∆Hcal/∆HvH

5.09 1.06 0.53 5.07 1.01 5.18 1.04 5.29 1.03 5.07 1.02 5.02 1.02 4.92 1.02 5.20 1.04 5.34 1.02

9.48 9.18 8.78 20.14 19.38 31.96 32.57 42.07 42.08 44.57 44.6 42.31 42.31 39.06 38.82 38.14 37.00 33.81 32.92

3.29 3.32 5.27 5.87 7.00 6.68 7.46 7.37 6.99 6.89 6.96 6.58 6.38 6.18 2.52 2.46

5378.0 5426.2 8620.0 9605.4 11447.4 10929.7 12198.7 12053.0 11435.3 11270.3 11372.6 10755.6 10425.1 10109.1 4125.7 4016.0

396.8 398.8 605.4 680.8 754.7 754.7 766.2 763.1 661.8 671.6 563.7 525.4 515.0 510.9 554.6 507.7

13.55 13.61 14.24 14.11 15.17 14.48 15.92 15.80 17.28 16.78 20.18 20.47 20.24 19.79

was in a dominant position to compete with water to interact with the hydroxyl of polysaccharide. Thus, with the continuous addition of DMSO, the hydrogen bonding network between the side-chain glucose residues and water molecules was broken, resulting in a decrease of Tm1. Interestingly, the Tm1 values for different lentinan concentrations (1.0 and 5.0 mg/mL) hardly changed, indicating that Tm1 is independent of the polysaccharide concentration in this range. The transition can be expressed as two states in character, which obeys the van’t Hoff equation34,36–38

enthalpy as a function of Tm1 is a straight line, independent of the sample concentration and the solvent composition, except for the datum at wDMSO ) 0.8495. When wDMSO ) 0.85, lentinan has already changed from triple helix to single random coils,24 resulting in the variation of ∆Hcal. On the basis of the data in Figure 6, the ∆Hcal-Tm1 relation in the lower temperature region can be expressed as follows:

∂ ln K ∆HvH ≡ ∂T RT2

The results in Table 2 and Figure 6 indicated that the binding energy of hydrogen bonds for the transition from triple helix I to II of lentinan is relatively low. Moreover, when wDMSO < 0.7, DMSO only surrounds the outside to protect the associating water layer, leading to the shift of Tm1 to high temperature. Thus, the energy to destruct the hydrogen bonds is also increased. When wDMSO was higher than 0.7, with the continuous addition of DMSO, the hydrogen bonding network between the sidechain glucose residues and water molecules was broken, resulting in a decrease of Tm1, corresponding to the decrease of the energy to destruct the hydrogen bonds. Thus, the slope of ∆Hcal vs Tm1 is positive. We can evaluate the cooperativity of the conformation transition for lentinan in the lower temperature region by means

(4)

where K ) θ/(1 - θ) is the equilibrium constant for the transition, θ is the molar number of cooperative units in one of the two states (such as the triple helix I or triple helix II), and ∆HvH is the effective enthalpy (i.e., the van’t Hoff enthalpy) that represents the heat absorbed due to conversion of 1 mol of cooperative units. In the middle of the transition, T ) Tm and θ ) 1/2; thus,

∆HvH ) 4RT2

Cp(Tm) Qtr

(5)

1 ∆Hcal ) 1036.6 + 248.7Tm

(7)

where Cp(Tm) is the maximal excess specific heat capacity of macromolecules, which can be obtained from the height of the endothermic peak in the US-DSC curve. Qtr is the total heat absorbed of the transition reaction for 1 g of polymer, which can be calculated by planimeter integration of the US-DSC curve. On the other hand, the “real” calorimetric enthalpy (∆Hcal) associated with the conversion of 1 mol of the whole polymer molecule can be calculated by

∆Hcal ) M · Qtr

(6)

where M is the molecular weight. The thermodynamic parameters for lentinan in the DMSO/water mixtures in the lower temperature region are summarized in Table 2. The ∆Hcal values of the lentinan solutions in the lower temperature region are plotted against Tm1, as shown in Figure 6. Clearly, the transition

Figure 6. Enthalpy change (∆Hcal) of the transition of lentinan as a function of the temperature of maximal excess specific heat capacity (Tm1) in the low temperature region.

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Figure 7. US-DSC curves of lentinan in a water-DMSO mixture at wDMSO ) 0.7799. The sample was heated up to 60 °C (----), and then reheated up to 130 °C after being cooled at 5 °C for 1 h. The second heating (s), the third heating ( · · · · ), and the forth heating (- · - · ).

Figure 8. Dependence of T02 for lentinan in DMSO/water mixtures on wDMSO in the high temperature region. Polysaccharide concentration: (O) c ≈ 5.0 mg/mL; (4) c ≈ 1.0 mg/mL.

of the ratio of ∆Hcal to ∆HvH (∆Hcal/∆HvH). Usually, in the case of the simplest type of two-state process which undergoes an all-or-none transition, the cooperative unit encompasses the whole molecule and therefore the effective enthalpy equals the real enthalpy, that is, ∆Hcal/∆HvH ) 1.34,41–43 However, for high molecular weight macromolecules, the conformation transitions are not all-or-none processes (not as a whole) as a result of the existence of the energy transfer process. This is a general phenomenon even for the synthetical homopolymers with simplex structure.34 By using protein terminology, it means that even a homopolymer can consist of a number of “melting domains”. The values of ∆Hcal/∆HvH of the conformation transition in the lower temperature region for lentinan in the DMSO/water mixtures are summarized in Table 2. The ratio ∆Hcal/∆HvH represents an effective number of cooperative units per polymer molecule, and it provides a quantitative measure of the degree of cooperativity.34,41–43 Therefore, when a conformation underwent a transition in the low temperature region, the lentinan sample consisted of cooperative units each with a molecular weight of about (7-13) × 104, corresponding to about 60-120 repeating units. This result reflects that the cooperativity of lentinan in the T1 region is poor, as a result of the diversity of the hydrogen bonding including intra- and intermolecular hydrogen bonds of lentinan, the intermolecular hydrogen bonding of the lentinan molecules with solvents, and the interaction between the side chains of lentinan and DMSO or water molecules. Moreover, the ratio of ∆Hcal/∆HvH slowly increased with increasing DMSO content in the solvent; that is, the cooperative unit size decreased. This suggests that the addition of DMSO enhanced the solvation of the mixed solvent, increasing the tendency of the formation of smaller cooperative units. The result further supports the existence of various hydrogen bonds, which induced multiple conformation transitions in lentinan. Figure 7 shows the cycling scanning US-DSC curves for transitions of the lentinan solution at wDMSO ) 0.7799. The sample solution was first scanned by heating from 5 to 60 °C through the transition at the T1 region and then rescanned after being cooled to 5 °C. It is noted that the lentinan sample underwent the same transition in the reheating process. The endothermic curve of the second heating scanning was nearly identical to that of the first scanning, suggesting that the transition for lentinan at lower temperature from triple helix I to triple helix II is reversible. This further confirmed that the energy to form the associating water layer by the self-assembly of water clusters and side groups of lentinan is relatively low and this is a dynamic process.

Conformation Transition at Higher Temperature. As shown in Figure 2, the second conformation transition of lentinan in the DMSO/water mixtures occurred at the higher temperature region (T2). When the temperature was raised to 130-145 °C, the triple-strand helical conformation of lentinan in aqueous solution changed to the single random coils.27 Thus, the endothermic peak in the higher temperature region reflects the dissociation of the helix structure. Therefore, the triple helix-single flexible chain transition occurred at higher temperature from 90 to 140 °C, depending to the DMSO concentration, namely, wDMSO from 0.8495 to 0. In the higher temperature region, the ability to destruct the hydrogen bonds of the elevated temperature in the second conformation transition of lentinan is equivalent to that of increasing DMSO concentration. The maximum detection temperature of the instrument is 130 °C, and the conformation transition temperature of lentinan exceeds the limit in a wide range of wDMSO values. The US-DSC curves are therefore not completed. Thus, the initial temperature of the transition (i.e., the beginning of the peak temperature, T02) is plotted against wDMSO. Figure 8 shows the dependence of T02 in the high temperature region on wDMSO for the lentinan solutions with two different polysaccharide concentrations (c ) 1.0 and 5.0 mg/mL). In the second transition region, the T02 values decrease with the addition of DMSO; namely, the endothermic peaks shift to lower temperature, suggesting that DMSO mainly contributes to destroy the intra- and intermolecular hydrogen bonds of lentinan in the high temperature region. When wDMSO was less than 0.7, T02 decreased slowly with increasing wDMSO, whereas, when wDMSO was more than 0.7, T02 decreased sharply with the continuous addition of DMSO. This suggests that the intra- and intermolecular hydrogen bonds were broken only at a relatively high energy region, and the two kinds of destruction occur simultaneously, because the energy needed was similar (in a narrow range). The two curves of lentinan at different polysaccharide concentrations nearly coincided with each other, indicating this change reflects the intrinsic characteristic of the structure of polysaccharide macromolecules. Furthermore, when wDMSO < 0.7, the thermal energy available at 110-120 °C was not sufficient to destroy the triple helix of lentinan. Only in the presence of a large amount of DMSO (wDMSO > 0.7), DMSO combined with the hydroxyl groups of lentinan to form new hydrogen bonds, leading to the destruction of the original hydrogen bonds of lentinan which sustain the triple helical structure. Figure 9 shows the temperature dependence of the specific heat capacity (Cp) of the lentinan solution at wDMSO ) 0.7799 with a polysaccharide concentration of 5.02 × 10-3 g/mL in the higher temperature region. Raising the temperature led to

Triple Helical Lentinan

J. Phys. Chem. B, Vol. 113, No. 29, 2009 9921

∆HvH ) 8RT2

Cp(Tm) Qtr

(9)

where T ) Tm2 and Tm2 is the temperature of maximal excess specific heat capacity in the high temperature region. From eq 9, we calculated the values of partial calorimetric parameters of the conformational transition for lentinan in the DMSO/water mixtures in the T2 region. The results are summarized in Table 3. Moreover, the ∆Hcal values for the T2 region are plotted against Tm2, as shown in Figure 10. The value of ∆Hcal increased linearly with elevating Tm2, showing a straight line. The ∆Hcal-Tm2 relation is described as Figure 9. Temperature dependence of the specific heat capacity (Cp) of lentinan in DMSO/water with wDMSO ) 0.7799 in the high temperature region detected by VP-DSC.

the increase of the polysaccharide chain motion, resulting in a downward trend for Cp(T) before the second transition (T2). When the temperature was raised to 98.4 °C, the molecular chain of lentinan dissociated from triple-strand helix to single random coils, leading to the rapid increase of Cp(T). This further confirms that the majority of the -OH groups in the lentinan were restricted by the strong intra- and intermolecular hydrogen bonds.21 Interestingly, when the triple helical lentinan changes to the single random coil conformation, the bounds of hydrogen bonds break, but the value of Cp is still larger than that before the transition. The main cause for these observations is believed to be due to an increase in the exposure of the single chain of lentinan to the surrounding solvent molecules, resulting in a sharp increase of the specific heat capacity for a system. The result from thermodynamics further confirmed that the transition of lentinan from triple helix to single random coils occurred at high temperature. The endothermic peaks of the transition for lentinan in the high temperature region exhibited clear asymmetry, and the simple dissociation reaction can be expressed as follows:36

(8)

An S nB

Here, A and B represent multistranded and single-chain molecules, respectively. n is the number of strands. If aggregation effects in the melting process are not taken into account, n ) 3 for the lentinan sample. To calculate the effective enthalpy of the reaction, eq 5 must be rewritten as

∆Hcal ) -7030.2 + 188.5Tm2

(10)

∆Hcal or Qtr represents the absorbed heat energy of transition for 1 mol or 1 g of lentinan, respectively. From the analysis of the results in Table 3 and Figure 10, the energy of the transition in the T2 region is much higher than that in the T1 region; i.e., the energy required for the breaking of the intra- and intermolecular hydrogen bonds of lentinan is higher than the binding energy of hydrogen bonds between its side group and water clusters. This indicated that the hydrogen bonding strength in the triple-stranded helix itself was relatively strong. On the basis of the ratio of ∆Hcal to ∆HvH, the number of cooperative units for the lentinan solutions in the high region was about 7-10. This indicated that the process during the transition from triple helix to single flexible chains for lentinan proceeded in a highly cooperative manner, suggesting the intra- and intermolecular hydrogen bonds are almost broken simultaneously. Furthermore, as shown in Figure 7, the dissociation of helical chain for lentinan at high temperature was irreversible. When the lentinan solution that had been heated through the transition at sufficiently high temperature was rescanned after being cooled to 5 °C, several endothermic peaks were observed in the USDSC curve. The result suggested that two or more single-strand chains of lentinan formed dimers, trimers, or higher aggregates by hydrogen bonds. In our previous work, the intrinsic viscosity of the denatured single-strand chain of lentinan in the DMSO/ water mixture was shown to be unable to reestablish the characteristic of the triple helical conformation. The intrinsic viscosity only increased slightly when water was added immediately to the solvent.24 Therefore, the different endothermic

TABLE 3: Calorimetric Parameters of the Transition at Higher Temperature for Lentinan in DMSO/Water Mixtures wDMSO 0.0 0.1998 0.3998 0.6004 0.6997 0.7799 0.8116 0.8299 0.8495

c (mg/mL)

T02 (°C)

5.09 1.06 5.07 1.01 5.18 1.04 5.29 1.03 5.07 1.02 5.02 1.02 4.92 1.02 5.20 1.04 5.34 1.02

122.66 121.02 118.94 117.89 118.17 117.14 117.02 116.96 113.70 116.88 98.35 99.71 87.65 89.02 80.03 81.33 72.29 71.48

Tm2 (°C)

Qtr (Jg1-)

∆Hcal (kJ mol-1)

∆HvH (kJ mol-1)

∆Hcal/∆HvH

125.31 127.19 111.38 113.4 100.24 103.91 98.73 99.35

10.14 10.34 8.31 8.54 7.63 8.25 9.29 9.20

16574.9 16908.0 13592.9 13967.6 12483.0 13493.9 15191.0 15046.4

2320.6 2200.1 1554.6 1682.2 1376.3 1495.7 1506.0 1531.8

7.14 7.69 8.74 8.30 9.07 9.02 10.09 9.82

9922

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Figure 10. Enthalpy change (∆Hcal) of the transition of lentinan as a function of the temperature of maximal excess specific heat capacity (Tm2) in the high temperature region.

Figure 11. Phase diagram for lentinan in DMSO/water mixtures.

peaks corresponded to the multiplicity of the chain structure of lentinan. Interestingly, the third heating scanning of the sample solution that had previously been heated twice gave almost the same US-DSC curve as that for the second heating, indicating that the denatured lentinan samples can form new aggregates by self-assembly and the aggregates undergo reversible transitions to single chains with temperature. The Intervals of Conformation Transition and Interaction of Hydrogen Bonds. Figure 11 shows the phase diagram of two kinds of conformational transitions for the lentinan solution induced by changes in temperature and in DMSO concentration. The triple helical structure of lentinan in aqueous solution can

Wang et al. be disrupted by heating or by strong solvents such as DMSO, which is similar to schizophyllan.44,45 Lentinan existed as the triple helix I in the DMSO/water mixtures (wDMSO ) 0-0.85) at 0-45 °C, corresponding to the A region, and as single flexible chains in DMSO (wDMSO ) 1), corresponding to the C region in the phase diagram. Lentinan in the aqueous solution underwent dissociation from triple-stranded helical chain to single random coils when the temperature was higher than 130 °C27 or when wDMSO g 0.85.24 Lentinan has been shown to change from triple helix I to triple helix II in pure water at temperatures lower than 10 °C, i.e., cross the phase boundary between region A and region B. More recently, we have found that the storage modulus G′ of lentinan in aqueous solution at 7-9 °C has shown a sharp decrease in hydrogen bonding, indicating that a well-organized triple helical structure was broken to a relatively disordered triple helical structure.46 In our findings, the conformational transition below 10 °C gave strong evidence that the internal change of the triple helix appears at the first interval from the A to the B region. The driving force for the change from the helix I to II of lentinan in the aqueous solution needs relatively low energy, whereas that from triple-strand helix to single flexible chains needs higher energy. In the phase diagram, the triple helical phase region occupies the main position, which further suggests that strong hydrogen bonding in lentinan dominates this system. In the A region, the hydrogen bond interactions between the side-chain glucose residues and the surrounding water clusters in the solution maintain a well-organized structure of triple helix I. The hydrogen bonds for the associating water network are weaker, so they are easily broken as the temperature rises slightly, leading to the change from the A to the B region. With the continuous addition of DMSO to reach higher wDMSO, the DMSO interaction is dominant compared with water clusters in the higher temperature region, promoting the formation of the new hydrogen bonds, leading to the destruction of the strong intra- and intermolecular hydrogen bonds in lentinan. Therefore, the diversity of the hydrogen bonds in the lentinan solutions induces the multiple conformation transitions. On the basis of the above results, a schematic diagram to describe the multiple conformational transition and hydrogen bonding interaction of lentinan in the aqueous solution is proposed in Figure 12. Before the first transition (region A), the hydroxyl in the side-chain glucose residues is combined with water clusters to form an associating water layer; it is assigned to “structured” water.37 In this case, the backbone of the triple helix is highly

Figure 12. Schematic diagram of a model describing the conformational transitions of lentinan in solution: (A) triple helical chain I; (B) triple helical chain II; (C) single flexible chain.

Triple Helical Lentinan immobilized by binding with the associating water and its side chain. At slightly elevated temperature, the triple helix I of lentinan is broken to form triple helix II due to the relatively low energy to destroy the associating water layer. Thus, the transition from the A region to the B region occurs. However, only when the temperature or wDMSO is very high (above 130 °C or wDMSO > 0.85, respectively), the second conformational transition from region B to C occurs, as a result of the destruction of the strong hydrogen bonding. This indicates that, once the energy is high enough, the intra- and intermolecular hydrogen bonds are almost simultaneously broken. In our previous work,27 both the distribution of Mw and Rh with temperature from SEC-MALLS and dynamic light scattering (DLS) showed two peaks, corresponding to the triple helical conformation and single-strand flexible chain, respectively. This also supported that the intra- and intermolecular hydrogen bonds of triple helical lentinan were simultaneously destroyed at 130-145 °C (i.e., the conformation transition interval at higher temperature). Conclusions There were two conformational transitions in the lentinan solution: one in the temperature range of 8-45 °C (T1), corresponding to the reversible transition from triple helix I to triple helix II, and the other at 90-140 °C (T2), corresponding to the irreversible conformation transition from triple-strand helix to single flexible chain. In the first transition region, the side chain of triple helix I was combined with water clusters through hydrogen bonds to form an associating water layer, leading to high immobilization of the backbone. However, the backbone and side chain could rotate more freely in triple helix II, as a result of the breaking of the associating water layer, which needed relatively low energy. In the second transition region, the intra- and intermolecular hydrogen bonds in lentinan were broken simultaneously, leading to the transition from triple helix chains to single flexible chains. The second transition needed higher energy, and therefore, it occurred at elevated temperature from 90 to 140 °C, depending on the DMSO concentration (wDMSO from 0.8495 to 0). When wDMSO was sufficiently high (>0.7), the main contribution of DMSO was to form new and relatively stable hydrogen bonds with lentinan, leading to the destruction of the original hydrogen bonds which maintain the helical conformation. Thus, when wDMSO > 0.7, the elevating temperature or increasing wDMSO promoted the second conformation transition of lentinan. The effects of raising the temperature and increasing the DMSO concentration were similar. Furthermore, the transition in the high temperature region showed high cooperation. Therefore, the diversity of the hydrogen bonding pattern in the lentinan aqueous solution induced the multiple conformation transitions for the triple helix polysaccharide. Acknowledgment. We gratefully acknowledge the major grant of the National Natural Science Foundation of China (30530850), the National Natural Science Foundation (20874079), and the High-Technology Research and Development Program of China (2006AA02Z102). References and Notes (1) 65–96. (2) (3) J. Biol.

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