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.     

Thermally induced conformation change of succinoglycan in aqueous sodium chloride

Static and dynamic light scattering, viscosity, and optical rotation measurements have been made at eight different temperatures between 25 and 75 degrees C on two succinoglycan samples (sodium salt) with weight-average molecular weights M(w) of 7.14 x 10(5) and 3.54 x 10(5) (at 25 degrees C) in 0.01 M aqueous NaCl to investigate the thermally induced order-disorder conformation change of the polysaccharide. Additionally, viscometry and polarimetry have been performed for a sodium salt sample (M(w) = 4.55 x 10(5) at 25 degrees C) whose M(w), z-average radius of gyration (z)(1/2), and hydrodynamic radius R(H) in the aqueous salt had been determined previously. As the temperature increases, M(w), (z)(1/2), R(H), and the intrinsic viscosity for every sample sharply decrease around 55 degrees C where the specific rotation at 300 nm sigmoidally increases. In particular, M(w) at 25 degrees C (i.e., in the ordered helical state) is twice as large as that at 75 degrees C (i.e., in the disordered state). These findings substantiate that the ordered structure is composed of two chains and hence is a double helix. Data analysis shows that this helix at 25 degrees C is characterized by an unperturbed wormlike chain with a helix pitch of about 2 nm (per repeating unit) and a persistence length of about 50 nm and that upon heating, it dissociates directly (i.e., in all-or-none fashion) to disordered chains of a similar contour length but with a much smaller persistence length of about 10 nm. The temperature dependence of the light scattering second viral coefficient is discussed in relation to the association of disordered chains in the cooling process.     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

A study of the chain stiffness and extension of alginates, in vitro epimerized alginates, and periodate-oxidized alginates using size-exclusion chromatography combined with light scattering and viscosity detectors

A series of alginates isolated from the stem and leaf of a brown algae (Laminaria hyperborea), bacterial mannuronan, in vitro epimerized mannuronans, and periodate oxidized alginates were analyzed by size-exclusion chromatography (SEC) combined with online multiangle laser light scattering (MALS) and viscometry (collectively abbreviated SMV). Selected samples were also analyzed off-line using low-angle laser light scattering and capillary viscometry. Excellent agreement between the two methods was obtained for properly purified samples. In contrast, abnormal results were obtained for some industrial samples due to the presence of particulate material. Naturally occurring alginates and in vitro epimerized mannuronans were found to obey essentially the same RG-M and [eta]-M relations, and hence, the same Mark-Houwink-Sakurada (MHS) equations (valid for I = 0.10 M): 20 000 g/mol < M < 100 000 g/mol, [eta] = 0.0054 .M(1.00); 100 000 g/mol < M < 1 000 000 g/mol, [eta] = 0.071 .M(0.89). Application of the wormlike chain model to the [eta]-M data obtained by SMV yielded persistence lengths (q) of 15 nm for all alginates at an ionic strength of 0.17 M. Intrinsic viscosities corresponding to infinite ionic strength were estimated on the basis of Smidsr?d's B-parameter, and the wormlike chain model then yielded q = 12 nm. Periodate oxidized alginates showed, in contrast, a pronounced decrease in persistence length with increasing degree of oxidation, reaching values below 4 nm at 44% oxidation. Periodate oxidation also resulted in some depolymerization, even in the presence of a free-radical scavenger.     

Small-angle neutron scattering study of the structure of protein/detergent complexes

Small-angle neutron scattering (SANS) was used to study the structure of protein/sodium dodecylsulfate complexes. Two water soluble proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were used. The protein concentration was kept constant at 1 wt %, and protein/detergent wt ratio varied between 1/1, 1/1.5, 1/2 and 1/3. Absolute intensities of SANS distributions were analyzed by a fractal model. Analyses of large Q portions of SANS distributions established that sodium dodecylsulfate (SDS) molecules bound to a protein/SDS complex form micelle-like clusters. On the other hand, analyses of small Q portions of SANS distributions clearly showed that the arrangement of micelle-like clusters resembles a fractal packing of spheres. We showed that a protein/SDS complex can be characterized by four parameters extracted from the scattering experiment, namely, the average micelle size and its aggregation number, the fractal dimension characterizing the conformation of the micellar chains, the correlation length giving the extent of the unfolded polypeptide chains, and the numbers of micelle-like clusters in the complex.     

Probing macromolecular architectures of nanosized cyclic structures of (1-->3)-beta-D-glucans by AFM and SEC-MALLS

Comb-like branched (1-->3)-beta-D-glucans dissolve in water as stiff triple-helical structures. Dissociation followed by re-association leads to the formation of a blend of various macromolecular topologies, where the cyclic species make up a significant fraction. In this study, the molecular properties of these nanosized cyclic structures of (1-->3)-beta-D-glucans were probed using a combination of AFM and SEC-MALLS. The cyclic structures were obtained by subjecting linear triple-helical molecules of (1-->3)-beta-D-glucans to a denaturation-renaturation cycle, and the fraction of cyclic structures in the renatured sample was determined by AFM. Samples containing different known fractions of linear and circular molecules were studied by SEC with online multi-angle laser-light scattering and viscometric detectors. The molecular weight and the radius of gyration of the molecules eluting from the SEC column, as well as the concentration and the intrinsic viscosity, were determined simultaneously. By extrapolating the results to a situation of only circular species, the results allowed to determine the linear mass per unit length (M(L)) of not only the linear but also the circular morphologies of the (1-->3)-beta-D-glucans. The values obtained were M(L)=2140+/-180 g mol(-1)nm(-1) for the circular species and 2045+/-80 g mol(-1)nm(-1) for the linear species. This is the first direct determination of the M(L) parameter of the circular topology, and the results indicate that the reassociation of the individual chains yield a triplex structure also for the circular morphology, similar to the initial triple helix.     

Complexation of lysozyme with poly(sodium(sulfamate-carboxylate)isoprene)

The complexation between hen egg white lysozyme (HEWL) and a novel pH-sensitive and intrinsically hydrophobic polyelectrolyte poly(sodium(sulfamate-carboxylate)isoprene) (SCPI), was investigated by means of dynamic, static, and electrophoretic light scattering and isothermal titration calorimetry measurements. The complexation process was studied at both pH 7 and 3 (high and low charge density of the SCPI, respectively) and under low ionic strength conditions for two polyelectrolyte samples of different molecular weights. The solution behavior, structure, and effective charge of the formed complexes proved to be dependent on the pH, the [-]/[+] charge ratio, and the molecular weight of the polyelectrolyte. Increasing the ionic strength of the solution led to vast aggregation and eventually precipitation of the complexes. The interaction between HEWL and SCPI was found to be mainly electrostatic, associated with an exothermic enthalpy change. The structural investigation of the complexed protein by fluorescence, infrared, circular dichroism spectroscopic, and differential scanning calorimetric measurements revealed no signs of denaturation upon complexation.     

Chain-length dependence of the kinetics of the hyaluronan hydrolysis catalyzed by bovine testicular hyaluronidase

Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8x10(2) g mol(-1) to 2.5x10(5) g mol(-1) and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme-substrate complex is maximum for HA molar masses ranging from 3x10(3) to 2x10(4) g mol(-1). Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2x10(4) g mol(-1), are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.     

The conformation of the poly(ethylene glycol) chain in mono-PEGylated lysozyme and mono-PEGylated human growth hormone

Covalent conjugation of poly(ethylene glycol) or "PEGylation" has proven an effective strategy to improve pharmaceutical protein efficacy by hindering recognition by proteases, inhibitors, and antibodies and by retarding renal clearance. Because it determines the strength and range of intermolecular steric forces and the hydrodynamic properties of the conjugates, the configuration of protein-conjugated PEG chains is the key factor determining how PEGylation alters protein in vivo circulation time. Mono-PEGylated proteins are typically described as having a protective PEG shroud wrapped around the protein, but recent dynamic light scattering studies suggested that conjugates adopt a dumbbell configuration, with a relatively unperturbed PEG random coil adjacent to the globular protein. We used small-angle neutron scattering (SANS) to distinguish between the dumbbell model and the shroud model for chicken-egg lysozyme and human growth hormone covalently conjugated to a single 20 kDa PEG chain. The SANS contrast variation technique was used to isolate the PEG portion of the conjugate. Scattering intensity profiles were well described by the dumbbell model and inconsistent with the shroud model.     

Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions)

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.     

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.     

Binding of hyaluronan to lysozyme at various pHs and salt concentrations.

Binding of hyaluronan (HA) to lysozyme immobilized on Sepharose-6B was investigated as a function of pH and NaCl concentration. High affinity binding (Kd = 1.0-2.0 x 10(-8) M) was observed at pH 7.5 and at 10-50 mM NaCl; the number of moles of HA bound to lysozyme was twice as high at 30 mM NaCl as at 10 mM. No specific binding was observed at and above 100 mM NaCl. Binding was suppressed in the presence of chaotropic agents such as guanidinium chloride and urea. These results suggest that binding between HA and lysozyme can occur in the extracellular matrix where an electrolyte concentration as low as 50 mM could be expected due to ionic exclusion by the highly negative charge concentration arising from the polyanions present.     

Solution properties of viilian, the exopolysaccharide from Lactococcus lactis subsp. cremoris SBT 0495

The exopolysaccharide (EPS) "viilian" was isolated from a large-batch fermentation of Lactococcus lactis subsp. cremoris SBT 0495. After applying a newly developed purification procedure, pure viilian with a weight-averaged molar mass of 2.64 x 10(3) kg/mol was obtained in a yield of 0.6 g/L culture broth. The native EPS, as well as lower molar mass fractions obtained by sonication of the native polymer, were studied by capillary viscometry and size-exclusion chromatography (SEC) coupled to multiangle laser light scattering detection (MALLS). From the viscosity data at various ionic strengths, we extracted a Mark-Houwink-Kuhn-Sakurada exponent a = 0.79, and a Smidsrod B value of 0.03. By application of the Hearst, Bohdanecky, and Odijk models for stiff polymer coils, in connection to the experimental viscosity data, we established the characteristic ratio to be C(infinity) = 44 and the intrinsic persistence length q(0) = 11.5 nm. The rms radii of gyration predicted from each of the models were in good agreement with the experimental radii (e.g., (1/2)(w) = 162 nm for native viilian in 0.2M NaNO(3)), as determined by SEC-MALLS. In addition, the Odijk model predicts correct ionic strength-linear charge density dependence of the rms radius of gyration. From the combined viscosity and SEC-MALLS experiments we concluded that, in dilute aqueous solutions, viilian behaves as an intermediately stiff, random coil polyelectrolyte system.Copyright 2000 John Wiley & Sons, Inc.     

Association behavior of biotinylated and non-biotinylated poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate)

Biotinylated and non-biotinylated copolymers of poly(ethylene oxide) (PEO) and poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) were synthesized by the atom transfer radical polymerization technique. The chemical compositions of the copolymers as determined by NMR are represented by PEO(113)PDEAEMA(70) and biotin-PEO(104)PDEAEMA(93), respectively. The aggregation behavior of these polymers in aqueous solutions at different pHs and ionic strengths was studied using a combination of potentiometric titration, dynamic light scattering, static light scattering, and transmission electron microscopy. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers form micelles at high pH with hydrodynamic radii (R(h)) of about 19 and 23 nm, respectively. At low pH, the copolymers are dispersed as unimers in solution with R(h) values of about 6-7 nm. However, at a physiological salt concentration (c(s)) of about 0.16 M NaCl and a pH of 7-8, the copolymers form large loosely packed Gaussian chains, which were not present at the low c(s) of 0.001 M NaCl. The critical micelle concentrations (cmc's) and the cytotoxicities of the copolymers were investigated to determine a suitable polymer concentration range for future biological applications. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers possess identical cmc values of about 0.0023 mg/g, while the cytotoxicity test indicated that the copolymers are not toxic up to 0.05 mg/g (>83% cell survival at this concentration).     

Complexation of DNA with poly(methacryl oxyethyl trimethylammonium chloride) and Its poly(oxyethylene) grafted analogue

Intermolecular complexes of genomic polydisperse DNA with synthetic polycations have been studied. Two cationic polymers have been used, a homopolymer poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its analogue grafted with poly(oxyethylene). The amount of poly(oxyethylene) grafts in the copolymer was 15 mol % and Mw of the graft was 200 g/mol. Salmon DNA (sodium salt) was used. The average molecular weight (Mw) of DNA was 10.4 x 10(6) g/mol. Conductivity, pH, and dynamic light scattering studies were used to characterize the complexes. The size and shape of the polyelectrolyte complex particles have been studied as a function of the cation-to-anion ratio in aqueous solutions of varying ionic strengths. The polyelectrolyte complexes have extremely narrow size distributions taking into account the polydispersity of the polyelectrolytes studied. The poly(oxyethylene) grafts on PMOTAC promote the formation of small colloidally stabile complex particles. Addition of salt shifts the macroscopic phase separation toward lower polycation content; that is, complexes partly phase separate with the mixing ratios far from 1:1. Further addition of salt to the turbid, partly phase separated solution results in the dissociation of complexes and the polycation and DNA dissolve as individual chains.     

Analysis of monoPEGylated human galectin-2 by small-angle X-ray and neutron scattering: concentration dependence of PEG conformation in the conjugate

Protein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility, and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two monosubstituted subunits per hGal-2 homodimer. Small angle X-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties. SAXS data gave a constant radius of gyration (R(g) = 21.5 ?) for the conjugate at different concentrations and provided no evidence for an alteration of homodimeric structure or hGal-2 ellipsoidal shape upon PEGylation. In contrast, SANS data revealed a concentration dependence of R(g) for the conjugate, with the value decreasing from 31.5 ? at 2 mg/mL to 26 ? at 14 mg/mL (based on hGal-2 concentration). Scattering data have been successfully described by the model of the ellipsoidal homogeneous core (hGal-2) attached with polymer chains (PEG) at the surface. Evidently, the PEG conformation of the conjugate strongly depends on conjugate concentration and PEG's radius of gyration decreases from 24.5 to 15 ?. An excluded volume effect, arising from steric clashes between PEG molecules at high concentration, was quantified by estimating the second virial coefficient, A(2), of PEGylated hGal-2 from the SANS data. A positive value of A(2) (6.0 ± 0.4 × 10(-4) cm(3) mol g(-2)) indicates repulsive interactions between molecules, which are expected to protect the PEGylated protein against aggregation.     

Hyaluronidase activity is modulated by complexing with various polyelectrolytes including hyaluronan.

Hyaluronidase (HAase) plays an important role in the control of the size and concentration of hyaluronan (HA) chains, whose biological properties strongly depend on their length. Our previous studies of HA hydrolysis catalyzed by testicular HAase demonstrated that, whilst the substrate-dependence curve has a Michaelis-Menten shape with a 0.15 mol L(-1) ionic strength, at low ionic strength (5 mmol L(-1)), a strong decrease in the initial hydrolysis rate is observed at high substrate concentrations; the HA concentration for which the initial rate is maximum increases when the HAase concentration is increased. After examination of various hypotheses, we suggested that this could be explained by the ability of HA to form non-specific complexes with HAase, which thus becomes unable to catalyze HA hydrolysis. In order to verify this hypothesis, we first showed from turbidimetric measurements that HAase, like albumin, is able to form electrostatic complexes with HA. Albumin then was used as a non-catalytic protein able to compete with HAase for the formation of non-specific complexes with HA, allowing HAase to be free and catalytically active. The kinetic results showed that the HA-HAase non-specific complex inhibits HAase catalytic activity towards HA. Depending on the albumin concentration with respect to the HAase and HA concentrations, albumin can either remove this inhibition or induce another type of inhibition. Finally, the extent of such non-specific interactions between polyelectrolytes and proteins in HAase inhibition or activation, in particular under in vivo conditions, is discussed.     

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.