Temporary network formation of hyaluronate under a physiological condition. 1. Molecular-weight dependence

The dynamic shear moduli at various frequencies and stress growth after the sudden start of steady shear were measured for 1% HA (hyaluronic acid) solutions with different molecular weights. From the results of the zero shear viscosity, eta 0, the steady shear compliance, J 0e, and delta eta defined as delta eta = eta 0 - eta a (infinity), where eta a (infinity) is the apparent viscosity at the steady state, it was shown that the molecular mechanism of flow of 1% HA solutions was classified into four regions with respect to molecular weight: (I) the viscosity-average molecular weight Mv less than 35 X 10(4), HA chains are dispersed molecularly in solution; (II) 35 X 10(4) less than Mv less than 100 X 10(4), the polymer chains form a weak entanglement network observed only through eta 0; (III) 100 X 10(4) less than Mv less than 160 X 10(4), the network is strengthened with increasing molecular weight and becomes detectable through both eta 0 and J 0e and also through the overshoot phenomenon; and (IV) 160 X 10(4) less than Mv, the network is "saturated" or "completed" dynamically. This is the conjecture presented for the first time by the present work.     

Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA.

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.     

Solution properties of targacanthin (water-soluble part of gum tragacanth exudate from Astragalus gossypinus)

Solution properties of tragacanthin (the water-soluble part of gum tragacanth) were studied by gel permeation chromatography (GPC) combined with multi-angle light scattering and viscometry at 25 degrees C. Photon correlation spectroscopy was used to determine the hydrodynamic radius. Ultrasonic degradation was applied to obtain biopolymer fractions of different molecular weights. The dependence of intrinsic viscosity [eta] and radius of gyration (s2)z(1/2) on weight average molecular mass M(w) for this biopolymer were found to be [eta] = 9.077 x 10(-5) M(w)(0.87) (dL g(-1)) and (s2)z(1/2) in the range of M(w) from 1.8 x 10(5) to 1.6 x 10(6). The conformational parameters of tragacanthin were calculated to be 1111 nm for molar mass per unit contour length (M(L)), 26 nm for persistence length (q) and 1.87 ratio of R(g)/R(h). It was found that the Smidsr?d parameter B, the empirical stiffness parameter was 0.013, which is lower than that of several polysaccharides indicating the stiff backbone for tragacanthin. The rheological behavior of aqueous solutions of gum tragacanth and its insoluble and soluble fractions (bassorin and tragacanthin, respectively) were studied. For concentrations equal to 1%, at 25 degrees C and in the absence of salt, bassorin solution showed the highest viscosity and shear thinning behaviour. Power law and Williamson models were used to describe the rheological behaviour of bassorin and tragacanthin, respectively. Oscillatory shear experiments showed a gel like structure for the bassorin but for tragacanthin the oscillatory data were as would be expected for semi-dilute to concentrated solution of entangled, random coil polymers. NaCl changed the steady and oscillatory rheological properties of both fractions and in this way the final viscosity of bassorin was even less than tragacanthin. The calculated activation energy for bassorin and tragacanthin indicated a more rapid decrease in viscosity with temperature for tragacanthin. The plot of eta(sp,0) versus C[eta] revealed that the transition from dilute to semi-dilute regime occurs at C*[eta] = 2.82 for tragacanthin.     

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.     

Viscosity behaviour and persistence length of sodium xanthan in aqueous sodium chloride

Zero-shear-rate intrinsic viscosities [eta] of sodium xanthan in aqueous NACl at 25 degrees C were determined for five samples ranging in weight- average molecular weight from 2 x 10(5) to 4 x 10(6) at salt concentrations Cs between 0.005 and 1 M, at which the polysaccharide maintains its double-helical structure. The measured [eta] for every sample was almost independent of Cs, in contrast to usual observations on flexible polyelectrolytes. The persistence length q of sodium xanthan was determined as a function of Cs by use of the theory of Yamakawa et al. for [eta] of an unperturbed worm-like cylinder, and from its Cs dependence the intrinsic persistence length q(o) ( = q at infinite ionic strength) was estimated to be 106 nm. This q(o) value was roughly twice as large as that of double-stranded DNA, indicating a high intrinsic rigidity of the xanthan double helix. The electrostatic contribution ( = q - q(o)) to q was only about 10% even at the lowest Cs of 0.005 M. Thus, it was concluded that above Cs = 0.005 M, the double- helical structure of sodium xanthan is hardly stiffened by electrostatic interactions between charged groups.     

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.     

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.     

Molecular mass and chain conformation of carboxymethylated derivatives of beta-glucan from sclerotia of Pleurotus tuber-regium

Seven water-insoluble (1 --> 3)-beta-D-glucan fractions TM8-1 to TM8-7 with weight-average molecular mass M(w) ranged from 2.22 to 77.4 x 10(4) obtained from the sclerotia of Pleurotus tuber-regium were carboxymethylated to produce the water-soluble fractions CTM8-1 to CTM8-7 with M(w) ranged from 3.87 to 87.8 x 10(4). The degree of substitution (DS) of CTM8 fractions was analyzed by ir and elemental analysis (EA) to be 0.3-0.68. The M(w) and the intrinsic viscosity [eta] of the CTM8 fractions were measured by size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), MALLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The dependencies of [eta] and radius of gyration (z) (1/2) on M(w) for the CTM8 samples were found to be [eta] = (8.82 +/- 0.03) x 10(-3) M(w)(0.78 +/- 0.04) (cm(3) g(-1)) and (z) (1/2) = (3.09 +/- 0.05) x 10(-3) M(w)(0.75 +/- 0.06) (nm) in the M(w) range from 3.87 x 10(4) to 53.2 x 10(4). Based on current theories for wormlike chain model, the conformational parameters of the CTM8 were obtained to be 790 (nm(-1)) for M(L), 9.6 (nm) for q, which were higher than those of the native TM8 fractions, suggesting a more extended flexible chain of CTM8 in PBS. On the whole, the CTM8 fractions showed higher antitumor activity than their corresponding TM8 fractions. In view of data from molecular parameters and bioactivity, the antitumor activity of the CTM8 fractions may be correlated to its water solubility and relatively extended chain.     

Chemical structure and chain conformation of the water-insoluble glucan isolated from Pleurotus tuber-regium

A water-insoluble polysaccharide (TM8) was isolated from sclerotium of Pleurotus tuber-regium by extraction with 0.5M NaOH aqueous solutions at 120 degrees C. Its chemical structure was confirmed by infrared, high performance liquid chromatography, gas chromatography, and (13)C NMR in dimethylsulfoxide (DMSO) to be composed of beta-(1 --> 3)-D-glucan backbone chain linked with a branched glucose, one out of every three glycosyl units being substituted at C6 position. The glucan TM8 in DMSO was fractionated by nonsolvent addition method into ten fractions, and the solution properties were studied by size exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) and viscometry in DMSO at 30 degrees C. The dependencies of intrinsic viscosity [eta] and radius of gyration [(s(2)(1/2)(z-2)] on weight-average molecular mass M(w) for this glucan were found to be [eta] = (9.24 +/- 0.2) x 10(-2)M(w)(0.51 +/- 0.02) (cm(3)g(-1)) and [(s(2)(1/2)(z-2)] = (3.67 +/- 0.3) x 10(-2)M(w)(0.56 +/- 0.02) (nm) in the range of M(w) from 1.07 x 10(4) to 77.4 x 10(4). Based on current theories for a wormlike chain, the conformational parameters of the glucan TM8 were found to be 408 (nm(-1)) for M(L), 3.1 (nm) for q, and 16.8 for C(infinity), suggesting that the polysaccharide exists as a dense random-coil chain in DMSO, due to branched structure.     

Evaluation of radius of gyration and intrinsic viscosity molar mass dependence and stiffness of hyaluronan

Nine hyaluronan (HA) samples were fractionated by size-exclusion chromatography, and molar mass (M), radius of gyration (Rg), and intrinsic viscosity ([eta]) were measured in 0.15 M NaCl at 37 degrees C by on-line multiangle light scattering and viscometer detectors. Using such method, we investigated the Rg and [eta] molar mass dependence for HA over a very wide range of molar masses: M ranging from 4 x 10(4) to 5.5 x 10(6) g/mol. The Rg and the [eta] molar mass dependence found for HA showed a meaningful difference. The Rg = f(M) power law was substantially linear in the whole range of molar masses explored with a constant slope of 0.6. In contrast, the [eta] = f(M) power law (Mark-Houwink-Sakurada plot) showed a marked curve shape, and a linear regression over the whole range of molar masses does not make sense. Also the persistence length (stiffness) for HA was estimated. The persistence length derived by using both the Odijk's model (7.5 nm from Rg vs M data) and the Bohdanecky's plot (6.8 nm from [eta] vs M data) were quite similar. These persistence length values are congruent with a semistiff conformation of HA macromolecules.     

Effects of excluded volume and polydispersity on solution properties of lentinan in 0.1 M NaOH solution

Seven lentinan fractions of various weight-average molecular weights (M(w)), ranging from 1.45 x 10(5) to 1.13 x 10(6) g mol(-1) were investigated by static light scattering and viscometry in 0.1M NaOH solution at 25 degrees C. The intrinsic viscosity [eta] - M(w) and radius of gyration s(2)(z) (1/2) - M(w) relationships for lentinan in 0.1M NaOH solution were found to be represented by [eta] = 5.1 x 10(-3)M(w) (0.81) cm(3) g(-1) and s(2)(z) (1/2) = 2.3 x 10(-1)M(w) (0.58) nm, respectively. Focusing on the effects of the M(w) polydispersity with the Schulz-Zimm distribution function, the data of M(w), s(2)(z) (1/2), and [eta] was analyzed on the basis of the Yoshizaki-Nitta-Yamakawa theory for the unperturbed helical wormlike chain combined with the quasi-two-parameter (QTP) theory for excluded-volume effects. The persistence length, molecular weight per unit contour length, and the excluded-volume strength were determined roughly to be 6.2 nm, 980 nm(-1), and 0.1, respectively. Compared with the theoretical value calculated by the Monte Carlo model, the persistence length is longer than that of the single (1 --> 3)-beta-(D)-glucan chain. The results revealed that lentinan exists as single-stranded flexible chains in 0.1M NaOH solution with a certain degree of expansion due to the electrostatic repulsion from the interaction between the OH(-) anions and lentinan molecules.     

Preparation of 32P-labeled murine immune interferon and its binding to the mouse immune interferon receptor

Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P-labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hydrolysis of 32P-Mu-IFN-gamma or 32P-labeled human IFN-gamma leads to the release of [32P]phosphoserine but not phosphothreonine or phosphotyrosine. With 32P-Mu-IFN-gamma, we have demonstrated that there are 5 X 10(3) to 1.5 X 10(4) receptors per-cell on several murine cell lines of diverse origin and that the Kd at 24 degrees C for these cells is in the range of 1 X 10(-10) to 1 X 10(-9) M. Covalent binding of 32P-Mu-IFN-gamma to its receptor results in the formation of several specific high-molecular weight products, the major one of which has an apparent molecular weight of 90,000-100,000. If this represents a 1:1 complex of Mu-IFN-gamma and its receptor (or its binding subunit), the murine interferon gamma receptor has a molecular weight of 75,000-85,000.     

Solution properties of (1-->3)-alpha-D-glucan and its sulfated derivative from Poria cocos mycelia via fermentation tank

From Poria cocos mycelia yielded via a pilot scale facility-fermentation tank, a water-insoluble (1-->3)-alpha-D-glucan coded as Pi-PCM3-I was isolated by extraction with 0.5 M NaOH/0.01 M NaBH(4) aqueous solution. Nine fractions from F1 to F9 with a weight-average molecular mass (M(w)) range from 7.75 x 10(4) to 57.3 x 10(4) were prepared from the Pi-PCM3-I sample by a nonsolvent addition method. The fractions were reacted with chlorosulfonic acid-pyridine complex to product water-soluble sulfated derivatives coded as S1 to S8 with M(w) from 2.36 x 10(4) to 14.5 x 10(4) and degree of substitution (DS) of 0.86-1.38. M(w), z-average radius of gyration (s(2) (z) (1/2)), the second virial coefficient (A(2)), and the intrinsic viscosity ([eta]) of the native and sulfated Pi-PCM3-I were measured by laser light scattering (LLS), size-exclusion chromatography combined with LLS (SEC-LLS), and viscometry at 25 degrees C. The Mark-Houwink equations for Pi-PCM3-I in 0.25 M LiCl/dimethylsulfoxide (DMSO) (Me(2)SO) and for its sulfated derivative in 0.15 M NaCl aqueous solution at 25 degrees C were established to be [eta] = 1.33 x 10(-2) M(w) (0.75+/-0.01) (mL g(-1)) and [eta] = 1.46 x 10(-4) M(w) (1.13+/-0.01) (mL g(-1)), respectively. On the basis of theories for a wormlike cylinder model, the conformational parameters of the native and sulfated Pi-PCM3-I were calculated to be 760 +/- 50 and 1060 +/- 30 nm(-1) for the molar mass per unit contour length (M(L)), 6.3 +/- 0.5 and 13.1 +/- 1 nm for the persistence length (q), and 14.9 +/- 0.2 and 31.8 +/- 1 for the characteristic ratio (C( proportional, variant)), respectively. The results revealed that Pi-PCM3-I existed as an extended flexible chain in 0.25 M LiCl/Me(2)SO, and its sulfated derivative existed as a semistiff chain in 0.15 M NaCl aqueous solution. Furthermore, Pi-PCM3-I possessed similar structure and molecular parameters to wc-PCM3-I from a rotary shaker; this suggests promising industrialization of Poria cocos polysaccharides.     

Properties of fractionated chondroitin sulphate from ox nasal septa

1. Chondroitin sulphate was isolated from bovine nasal septa by precipitation with cetylpyridinium chloride after digestion of the tissue with papain. 2. The material was divided into two portions, one of which was partially degraded with testicular hyaluronidase. 3. Untreated and hyaluronidase-digested material were fractionated into a total of eleven subfractions by gel chromatography on Sephadex G-200 and Sephadex G-100 respectively. 4. Chemical analyses indicated that the composition of all the fractions was similar to that of chondroitin sulphate. However, electrophoresis revealed a charge-inhomogeneity in the low-molecular-weight fractions obtained after hyaluronidase digestion. 5. The physicochemical properties of the subfractions were investigated by sedimentation-velocity, diffusion and sedimentation-equilibrium studies, osmometry, viscometry and gel chromatography. The individual fractions were essentially monodisperse and showed molecular weights ranging from 2400 to 36000. 6. The relationship between the intrinsic viscosity and the molecular weight was [eta]=5.0x10(-6)xM(1.14), indicating that the chondroitin sulphate molecules assume a shape intermediate between that of a random coil and a stiff rod. 7. The relationship between the sedimentation constant and the molecular weight (>10(4)) was s(0) (20,w)=2.3x10(-2)xM(0.44).     

Molecular weight and related properties of lily amylose determined by monitoring of elution from TSK-GEL PW high performance gel chromatography columns by the low-angle laser light scattering technique and precision differential refractometry

Amylose was fractionated according to its molecular weight by high performance gel chromatography using columns of a TSK-GEL PW series. Elution from the columns was monitored with a low-angle laser light scattering photometer and a precision differential refractometer. The following results were obtained indicating that the procedure is highly efficient for characterizing an amylose preparation with respect to its molecular weight: 1) the weight-average molecular weight of lily amylose used as a test material was determined to be 786,000 +/- 26,000 (n = 7); 2) the molecular weight distribution curve of the amylose was worked out from the chromatographic data; and 3) based on the concept of the universal calibration curve, the Mark-Houwink-Sakurada equation of the amylose was presumed to be [eta] = 2.27 X 10(-4)M0.62 (dl/g). The technique saves time and sample significantly compared with the conventional ones, and consequently enables the characterization of amylose in aqueous solvents without either the degradation or association peculiar to the amylose molecule.     

Pressure cell assisted solution characterization of polysaccharides. 2. Locust bean gum and tara gum

Following the work carried out on guar gum in our first paper of a series, the "pressure cell" solubilization method was applied to two other less highly substituted galactomannans: locust bean gum (LBG) and tara gum. True molecular solution of the polymers was achieved using appropriate temperature, time, and pressure regimes. The technique of capillary viscometry was used to determine the intrinsic viscosity [eta] of the "pressure cell" treated and untreated samples. Molecular weight (M(w)) and radius of gyration (R(g)) were determined by light scattering. The data obtained for LBG and tara gum were compared statistically with reliable data found for guar gum in the literature. The variation in [eta] with M(w) followed the Mark-Houwink-Sakurada relationship, giving the exponent alpha = 0.74 +/- 0.01 for galactomannans consistent with random coil behavior. The characteristic ratio, C(infinity), and the chain persistence length, L(p), were both calculated for LBG and tara gum using the Burchard-Stockmayer-Fixman (BSF) method which is appropriate for flexible to semiflexible chains. A general value of 9 < C(infinity) < 16 and 3 < L(p) < 5 nm can now be estimated with statistical confidence for all galactomannans. According to our statistical analysis, the chain persistence length was found to be insensitive to the degree of galactose substitution.     

Rheological and light scattering properties of flaxseed polysaccharide aqueous solutions

Polysaccharides isolated from flaxseed meals using ethanol consisted of a soluble ( approximately 7.5% w/w) and an insoluble fraction (2% w/w). The soluble fraction was dialyzed in various salt concentrations and characterized using viscometry and light scattering techniques. Observations using a size-exclusion column coupled to a multiangle laser light scattering (SEC-MALLS) revealed three molecular weight fractions consisting of a small amount ( approximately 17%) of large molecular weight species (1.0 x 10(6)) and a large amount ( approximately 69%) of small molecular weight species (3.1 x 10(5) Da). Dynamic light scattering measurements indicated the presence of very small molecules (hydrodynamic radius approximately 10 nm) and a very large molecular species (hydrodynamic radius in excess of 100 nm); the latter were probably aggregates. The intrinsic viscosity, [eta], of the polysaccharide in Milli-Q water was 1030 +/- 20 mL/g. The viscosity was due largely to the large molecular weight species since viscosity is influenced by the hydrodynamic volume of molecules in solution. The Smidsrod parameter B obtained was approximately 0.018, indicating that the molecules adopted a semi-flexible conformation. This was also indicated by the slope ( approximately 0.56) from the plot of root-mean-square (RMS) radius versus molar mass (M(w)).     

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.     

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.     

Physicochemical properties of chondroitin sulfate. II. Molecular weight dependence of intrinsic viscosity and sedimentation coefficient, and analysis as a semi-flexible coil

1. The relation between intrinsic viscosity in 0.2 M NaCl and molecular weight for samples of chondroitin sulfate isolated from shark cartilage could be expressed by [eeta] = 6.5 X 10(-6) Mr114. 2. Their sedimentation coefficients were linear with respect to the square root of molecular weight, and the intercept was not zero but definitely positive, indicating that chondroitin sulfate in solution can be represented by a semi-flexible coil with a molecular diameter of about 1.2 nm. This value was consistent with the results of X-ray analysis. 3. The molecular weight dependence of the sedimentation coefficient for chondroitin sulfate from ox nasal septa cartilage was similar to that for chondroitin sulfate isolated from shark cartilage.