Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

The cytokine stimulating activity of (1-->3)-beta-D-glucans is dependent on the triple helix conformation

The immunomodulating properties of comb-like branched (1-->3)-beta-D-glucans scleroglucan, schizophyllan and lentinan depend on branching pattern, molecular weight and higher-order structure. The effect of weight average molecular weight Mw and higher order structure of scleroglucan, on stimulation of human monocytes cultured in vitro to secrete tumor necrosis factor-alpha (TNF-alpha) was investigated. The higher order structures of the scleroglucan samples were determined by electron microscopy. The data showed that the samples with a linear wormlike, triple helical structure with Mw less than 50 x 10(4) g/mol or larger than 110 x 10(4) g/mol stimulated the monocytes more efficiently than samples with Mw in the range (67-110) x 10(4) g/mol. The denaturation of the linear triple helices by NaOH (> 0.25 M), followed by neutralization yielded blends of linear and macrocyclic topologies with concomitant irreversible reduction of the cytokine inducing activity compared with the untreated scleroglucans. The dose-dependent ability to activate monocytes to cytokine production was not restored following annealing of the denatured-renatured samples, despite the fact that electron micrographs revealed similar structures of these annealed samples to the starting material. Pre-incubation of monocytes with antibodies against cluster of differentiation antigens CD14 or CD11b reduced the scleroglucan potency to stimulate TNF-alpha secretion mainly for mAb against CD14 in the presence of serum.     

Solution properties of antitumor sulfated derivative of alpha-(1-->3)-D-glucan from Ganoderma lucidum

Four fractions of a water-insoluble alpha-(1-->3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80 degrees C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and 13C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight Mw and intrinsic viscosity [eta] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The Mw value (2.4 x 10(4)) of sulfated glucan S-GL-1 was much lower than that (44.5 x 10(4)) of original alpha-(1-->3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C(infinity) for the S-GL in 0.2 M NaCl aqueous solution at 25 degrees C were found to be: [eta] = 1.32 x 10(-3) Mw(1.06) (cm3 g(-1)) and 16, respectively, in the Mw range from 1.1 x 10(4) to 2.4 x 10(4). It indicated that the sulfated derivatives of the alpha-(1-->3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the alpha-(1-->3)-D-glucan and curdlan, a beta-(1-->3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.     

Molecular mass and antitumor activities of sulfated derivatives of alpha-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1-->3)-alpha-D-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1-S5 and ac-PCM3-I-S1-S5, respectively. The derivatives having different weight-average molecular mass (Mw) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39-0.67 for ab-PCM3-I-S and 0.73-0.96 for ac-PCM3-I-S, respectively. The Mw and the intrinsic viscosity ([eta]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The results indicated that their Mw ranged from 2.0 to 11.3 x 10(4) for the samples ab-PCM3-I-S, and 4.7 to 40.0 x 10(4) for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native alpha-D-glucans. Therefore, a moderate range of molecular mass from 2.0 x 10(4) to 40.0 x 10(4), relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Melting behaviour of a triple helical polysaccharide schizophyllan in aqueous solution

Two sonicated samples of schizophyllan in aqueous solution at temperatures from 20 to 160°C were investigated by viscometry. The temperature dependence of the viscosity coefficient η showed that schizophyllan in water undergoes an irreversible thermal transition at about 135°C. The values of $\text{(ln η}{}_{\mathrm{r}}\text{)}\text{c}$ (ηr is the relative viscosity and c is the polymer concentration (w/v)) at 25°C determined after preheating aqueous schizophyllan indicated that the major conformations of schizophyllan in water at 120 and 150°C are triple helix and single random coil, respectively. Thus, it was concluded that the change in η at about 135°C with an increase in temperature is due to the melting of triple helices to single chains. Schizophyllan denatured to single chains at about 150°C did not restore the intact triple helix, but formed aggregates, when the solution was cooled to 25°C. It was also found that the aggregates form a gel when c is higher than a certain value.     

Physicochemical properties and antitumor activities of water-soluble native and sulfated hyperbranched mushroom polysaccharides

A water-soluble hyperbranched beta-glucan, coded as TM3b, extracted from sclerotia of an edible fungus (Pleurotus tuber-regium) was fractioned into eight fractions coded as F1-F8 by a nonsolvent addition method. Five fractions were treated with chlorosulfonic acid at 35 degrees C to synthesize successfully sulfated derivatives coded as S-F2, S-F3, S-F4, S-F5, and S-F8 with degree of substitution of 0.28-0.54. The 13C NMR results of these sulfated beta-glucans indicated that while the C-6 position was fully substituted, C-2, C-3, and C-4 were only partially substituted by the sulfate groups. The weight-average molecular weights (Mw) and intrinsic viscosities ([eta]) of the native and sulfated TM3b fractions were determined using multi-angle laser light scattering and viscometry in 0.15M aq NaCl at 25 degrees C, respectively. The dependences of [eta] on Mw for TM3b and sulfated TM3b were found to be [eta]=0.18Mw(0.28+/-0.03) (Mw range from 3.30 x 10(4) to 3.90 x 10(7)) and [eta]=2.24 x 10(-2)Mw(0.52+/-0.06) (Mw range from 3.24 x 10(4) to 3.15 x 10(5)) in 0.15M aq NaCl at 25 degrees C, respectively. It revealed that both the native TM3b and its sulfated derivatives exist in a spherical chain conformation in 0.15M aq NaCl. Furthermore, the native and sulfated TM3b fractions showed potent antitumor activities in vivo and in vitro. The sulfated derivatives exhibited relatively higher in vitro antitumor activity against human hepatic cancer cell line HepG2 than the native TM3b. Water solubility and introduction of sulfate groups were the main factors in enhancing the antitumor activities.     

Structure and chain conformation of beta-glucan isolated from Auricularia auricula-judae

From Auricularia auricula-judae, a water soluble beta-D-glucan, named as AAG, was isolated by extraction with 70% ethanol/water solution. Its chemical structure was analyzed by gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption /ionization (MALDI)-time of flight (TOF), and 1D, 2D NMR. AAG was detected, for the first time, to be composed of a main chain of (1-->4)-linked D-glucopyranosyl with glucopyranosyl side groups at O6. With the help of MALDI-TOF-MS, the sequence and the distribution of glucuronic acid were determined and the content of glucuronic acid is about 19%. Five fractions were prepared from the AAG sample in water by ultrasonic degradation method. Their molecular weight, size, and shape (chain conformation) were studied by dynamics light scattering (DLS), static laser light scattering (LLS), size exclusion chromatography combined LLS (SEC-LLS) and viscometry in 0.1M NaCl aqueous solution at 25 degrees C. The dependence of intrinsic viscosity ([eta]) on Mw for this polysaccharide was established to be [eta] = 1.22 x10(-3)Mw (1.00) (cm3 g(-1)) in the range of Mw from 3.40 x 10(4) to 2.88 x 10(5). The conformational parameters of the AAG polysaccharide were found to be 820 nm(-1) for molar mass per unit contour length (ML), 12.3 nm for persistence length (q) and 2.1 for rho (s2(1/2)/Rh). The results suggested that the polysaccharide exists as extended chains in 0.1M NaCl aqueous solution. The chemical structure of AAG containing glucuronic acid and side groups led to steric hindrance, resulting in the increased stiffness of the chains.     

Correlation between antitumor activity, molecular weight, and conformation of lentinan

A (1-->3)-beta-D-glucan having (1-->6) branching (L-FV-IB) from Lentinus edodes in water was degraded into seven fractions of different molecular weights by ultrasonic irradiation, and each was further fractionated into three parts, by precipitation from water into acetone at room temperature. The weight-average molecular weight (M(w)), radius of gyration ((z)(1/2)), and intrinsic viscosity ([eta]) of lentinan and its fractions in 0.9% NaCl aqueous solution and dimethyl sulfoxide (Me(2)SO) were determined by size-exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS), LLS, and viscometry. Analysis of M(w), [eta], and (z)(1/2) in terms of known theory for worm-like chains yielded 2240 +/- 100 nm(-1), and 100 +/- 10 nm for molar mass per unit contour length (M(L)), and persistence length (q), respectively, corresponding with theoretical data for triple-helical chains. The [alpha](D) of lentinan in water-Me(2)SO mixtures indicated an order-disorder transition. The results indicated that lentinan exists as a triple helix in 0.9% NaCl aqueous solution and as a single flexible chain in Me(2)SO. Assays in vivo and in vitro against the growth of Sarcoma 180 solid tumor as well as the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method for lentinan showed that the triple-helix sample exhibited a relatively high inhibition ratio. Interestingly, the triple-helix lentinan with M(w) of 1.49 x 10(6) exhibited the highest antitumor activity in vivo, having an inhibition ratio (xi) of 49.5%, close to that of 5-fluorouracil (xi = 50.5%), whereas the bioactivity (xi = 12.3%) of its single flexible chains almost disappeared. The triple-helix conformation plays an important role in enhancing the antitumor effects of lentinan.     

Effects of urea and sodium hydroxide on the molecular weight and conformation of alpha-(1-->3)-D-glucan from Lentinus edodes in aqueous solution

The weight-average molecular weight (Mw) and intrinsic viscosity ([eta]) of the alpha-(1-->3)-D-glucan (L-FV-II) from Lentinus edodes in 0.5 and 1.0 M NaOH aqueous solution containing urea, were studied by light scattering and viscometry. The Mw value of the glucan decreased with increase of the urea and NaOH concentration. A strong intermolecular hydrogen bonding confers water-insolubility on the glucan, but NaOH and especially urea, broke this hydrogen bonding leading to enhanced water-solubility. Use of 1.0 M urea-1.0 M NaOH as solvent broke not only intermolecular hydrogen bonds but also partial covalent bonds of the alpha-glucan in aqueous solution, resulting in a decrease of Mw and [eta]. The urea and NaOH concentrations, storage time with stirring, and mode of preparation of the polysaccharide in aqueous solution significantly affected the determination of Mw and [eta]. The dependences of specific rotation and fluorescence emission ratio of a probe on urea concentration showed that a change in the molecular conformation of the alpha-glucan in 0.5 M NaOH aqueous solution containing urea occurred in the range 0.4-0.6 M urea. The 0.5 M urea-0.5 M NaOH aqueous solution is a suitable solvent for the glucan, and the Mw and [eta] values obtained were 5.21 x 10(5) and 148 cm3 g(-1), respectively. Degradation of the glucan was obvious after storage for 15 months.     

Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. 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. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.     

Chain conformation of sulfated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Six water-insoluble (1-->3)-beta-D-glucan fractions TM8-1 to TM8-6 with weight-average molecular mass Mw ranging from 5.76 to 77.4x10(4) obtained from the sclerotia of Pleurotus tuber-regium were sulfated to produce the water-soluble fractions S-TM8-1 to S-TM8-6 with Mw from 6.0 to 64.8x10(4). The degree of substitution (DS) of S-TM8 fractions was analyzed by elemental analysis (EA) to be 1.14-1.74. The 13C NMR results indicated that the C-6 was fully substituted, and C-2, C-4 were partially substituted by the sulfo-groups. The Mw and the intrinsic viscosity [eta] of the S-TM8 fractions were measured, respectively, by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependences of [eta] and radius of gyration z(1/2) on Mw for the S-TM8 samples were found to be [eta]=1.89x10(-2) Mw(0.70) (cm3/g) and z(1/2)=1.12x10(-4) Mw(0.81) (nm) in the Mw range tested. Based on current theories for a wormlike chain model, the molar mass per unit contour length ML and persistence length q of the S-TM8 were calculated to be 990 nm(-1) and 8.5 nm, respectively. The relatively higher q value suggested a more expanded flexible chain of S-TM8 in PBS. The water-solubility and relatively expanded chain conformation of the STM8 fractions were considered to be significant to their antiviral activity.     

Antitumor activities of O-sulfonated derivatives of (1-->3)-alpha-D-glucan from different Lentinus edodes

Four water-insoluble (1-->3)-alpha-D-glucans, coded L-II1, L-II2, L-II3 and L-II4, with different molecular weights were isolated from four kinds of fruiting bodies of Lentinus Edodes. The four alpha-D-glucans were O-sulfonated to obtain derivatives (SL-II) having degrees of substitution (DS) from 0.9 to 2.1 respectively. The structure of the samples was analyzed by infrared spectra, elemental analysis, and 13C NMR. The weight-average molecular weight (Mw), radii of gyration (z1/2) and intrinsic viscosity ([eta]) of the native alpha-D-glucans and O-sulfonated derivatives were measured by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in 0.2 M aqueous NaCl and in dimethyl sulfoxide (DMSO) containing 0.25 M LiCl at 25 degrees C respectively. The Mw values of the O-sulfonated derivatives were much lower than those of the native alpha-D-glucans. The experimental results indicate that the O-sulfonated derivatives are water-soluble and exist as an expanded flexible chain in aqueous solution owing to intramolecular hydrogen bonding or interaction between charge groups. The in vivo and in vitro antitumor activities of the native alpha-D-glucans and their O-sulfonated derivatives against solid tumor Sarcoma 180 cells were evaluated and compared. Interestingly, all of the O-sulfonated derivatives exhibited higher antitumor activities than those of the native glucans. The results reveal that the effect of O-sulfonation of the alpha-D-glucan on the improvement of their antitumor activities was considerable.     

Solution properties of an alpha-(1-->3)-D-glucan from Lentinus edodes and its sulfated derivatives

A water-insoluble alpha-(1-->3)-D-glucan (A) from Lentinus edodes was fractionated into 13 fractions in dimethyl sulfoxide containing 0.25 M lithium chloride (0.25 M LiCl-Me(2)SO). Five fractions were treated with sulfur trioxide-pyridine complex at 25 degrees C to synthesize water-soluble sulfated derivatives (S-A). The weight-average molecular weights, M(w), and intrinsic viscosities [eta], of the samples A and S-A were determined by multi-angler laser light scattering (MALLS), and viscosity. The M(w) dependence of [eta] and of the radius of gyration (z)(1/2), was found to be represented approximately by [eta]=4.9 x 10(-2) M(w)(0.67) (cm(3) g(-1)), and (z)(1/2)=4.8 x 10(-2) M(w)(0.54) (nm) for the alpha-glucan in 0.25 M LiCl-Me(2)SO in the M(w) range from 7.24 x 10(4) to 4.21 x 10(5), and by [eta]=6.8 x 10(-4) M(w) 1.06 (cm(3) g(-1)), and (z)(1/2)=9.4 x 10(-4) M(w)(0.92) (nm) for the sulfated alpha-glucan in aqueous 0.5 M NaCl in the M(w) range from 5.92 x 10(4) to 1.42 x 10(5) at 25 degrees C. The results indicate that the alpha-(1-->3)-D-glucan exists as a flexible chain in 0.25 M LiCl-Me(2)SO, and its sulfated derivative in 0.5 M NaCl aqueous has stiffer chains than the original. (13)C NMR indicated that intramolecular hydrogen bonding occurred in the sulfated alpha-glucan, causing the observed chain stiffness.     

Gelation behaviors of schizophyllan-sorbitol aqueous solutions

On addition of D-sorbitol, schizophyllan (SPG) aqueous solution forms a thermoreversible gel upon cooling. The gelation process is characterized by rheology, differential scanning calorimetry (DSC), and optical rotation measurement (ORD). It is found that the Winter-Chambon criterion works well in determining the critical gelation point of the present system, although the criterion has been scarcely applicable to systems that show weak-gel properties even before gelation. Moreover, ORD and DSC results indicate that a disordered to ordered conformational change accompanies the gelation process, which is attributed to the transition from SPG triple helix II to I. The gelation temperature of SPG-sorbitol aqueous solution is almost independent of SPG concentration in the examined concentration range and is slightly decreased by lowering SPG molecular weight, while greatly influenced by sorbitol content. The gelation is considered to be induced by the transition from SPG triple helix II to I, which leads to a three-dimensional network constituted by the extremely entangled and stiff SPG triple helices I. Furthermore, it is proved that neither junction zone nor aggregation of SPG triple helices is involved in the SPG-sorbitol gels. The SPG-sorbitol gel is structurally like a solution that is unable to flow within a timescale of usual observation.     

Two types of rat gastric mucus glycoprotein subunits

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.     

Solution properties of chitin in alkali

The solution properties of alpha-chitin dissolved in 2.77 M NaOH are discussed. Chitin samples in the weight-average molecular weight range 0.1 x 10(6) g/mol to 1.2 x 10(6) g/mol were prepared by heterogeneous acid hydrolysis of chitin. Dilute solution properties were measured by viscometry and light scattering. From dynamic light scattering data, relative similar size distributions of the chitin samples were obtained, except for the most degraded sample, which contained aggregates. Second virial coefficients in the range 1 to 2 x 10(-3) mL.mol.g(-2) indicated that 2.77 M NaOH is a good solvent to chitin. The Mark-Houwink-Sakurada equation and the relationship between the z-average radius of gyration (Rg) and the weight-average molecular weight (Mw) were determined to be [eta] = 0.10Mw0.68 (mL.g(-1)) and Rg = 0.17Mw0.46 (nm), respectively, suggesting a random-coil structure for the chitin molecules in alkali conditions. These random-coil structures have Kuhn lengths in the range 23-26 nm.     

Phase separation in the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution driven by a specific interaction between the glucose side chain and poly(ethylene oxide)

We found that the mixture of schizophyllan and poly(ethylene oxide) in aqueous solution underwent phase separation at around 3-4 degrees C, and this temperature was independent of both polymer concentration and the difference in poly(ethylene oxide) molecular weight (Mw 6000 and 70,000). The phase-separation took place at the same temperature at which the optical rotation changed. Since the optical rotation change is ascribed to the difference in the nature of hydrogen bonding between the schizophyllan side chain and water, the phase separation is also considered to be due to an interaction between poly(ethylene oxide) and schizophyllan. The phase-separation temperature increased on changing H2O to D2O in accordance with a change in the optical rotation, confirming the specific interaction essential for the phase separation.     

Rheological properties and molecular structure of tunicate cellulose in LiCl/1,3-dimethyl-2-imidazolidinone

Solution properties and molecular structure of tunicate cellulose (TC), an animal cellulose from Halocynthia roretzi, were investigated in terms of rheological and dilute solution properties. The solvent used is 8 wt % LiCl/1,3-dimethyl-2-imidazolidinone (DMI). A solution of dissolving pulp (DP), derived from plant, was also used for comparison. The weight-average molecular weight, Mw, and the limiting viscosity number, [eta], of the TC were evaluated to be 413 x 10(6) and 2645 mL/g, respectively. The TC solution showed the same concentration dependence of GN (GN=5.49 x 10(6)phiw(2.1)4 Pa; phiw: weight fraction of cellulose in solution; GN: plateau modulus) as the DP solution and, moreover, also as the solution of cotton linter (CC) in 8 wt % LiCl/N,N-dimethylacetamide (DMAc). This exponent of 2.1(4) indicates that network structure by entanglements was formed in these solutions. According to the theory of Fetters et al., moreover, such identity means that all of these celluloses have the identical chain structure though their biological origins are far different. On the other hand, the phiw-dependence of eta0-etas (eta0=zero shear rate viscosity of solution; etas=solvent viscosity) was different between the TC and the DP solution in the semidilute regime: the TC solution exhibited eta0-etas proportional, variant phiw(7.5) and the DP solution eta0-etas proportional, variant phiw4. According to the theory of Doi-Edwards, this exponent of 4 (the DP solution) indicates that the DP behaves as flexible polymers in the solution. In contrast, the dependence for the TC solution seems unexplainable on the basis of molecular theories. This difference probably signifies the difference in the relaxation process or mechanism in entanglement systems.     

Chain conformation of water-insoluble hyperbranched polysaccharide from fungus

Water-insoluble polysaccharide (TM3a), extracted from sclerotia of Pleurotus tuber-regium, was identified as a hyperbranched beta-d-glucan from the results of one- and two-dimensional NMR and GC-MS analysis. The degree of branching of TM3a is 65.5%. TM3a was fractionated by using a non-solvent addition method into 14 fractions, and its solution properties in 0.25 M LiCl/dimethylsulfoxide (DMSO) solution were studied systematically by using static laser light scattering, dynamic light scattering, and viscometry at 25 degrees C. The dependences among the values of intrinsic viscosity ([eta]), radius of gyration (z 1/2), and hydradynamic radius (Rh) on weight-average molecular weight (Mw) were found as the following: [eta] = 0.46Mw0.30+/-0.01, z 1/2 = 4.79 x 10-2Mw0.43+/-0.04, and Rh = 5.01 x 10-2Mw0.41+/-0.02 in the Mw range from 1.94 x 105 to 2.06 x 107 for TM3a in a 0.25 M LiCl/DMSO solution at 25 degrees C. The current theory of polymer solution was applied to explain the relationship among the fractal dimension, ratio of geometric to hydrodynamic radius (rho = z 1/2/Rh), and MwA2/[eta] of TM3a. The results indicated that TM3a existed as a compact chain conformation with a sphere-like structure in LiCl/DMSO solution. Furthermore, by using transmission electron microscopy, we observed directly the spherical molecules with an average diameter of 23.0 +/- 1.8 nm.