Size Exclusion Chromatography Combined With Multi-angle Laser Light Scattering Research Articles

Probing Protein Folding with Sequence-Reversed α-Helical Bundles.

Recurrent protein folding motifs include various types of helical bundles formed by α-helices that supercoil around each other. While specific patterns of amino acid residues (heptad repeats) characterize the highly versatile folding motif of four-α-helical bundles, the significance of the polypeptide chain directionality is not sufficiently understood, although it determines sequence patterns, helical dipoles, and other parameters for the folding and oligomerization processes of bundles. To investigate directionality aspects in sequence-structure relationships, we reversed the amino acid sequences of two well-characterized, highly regular four-α-helical bundle proteins and studied the folding, oligomerization, and structural properties of the retro-proteins, using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography combined with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS). The comparison of the parent proteins with their retro-counterparts reveals that while the α-helical character of the parents is affected to varying degrees by sequence reversal, the folding states, oligomerization propensities, structural stabilities, and shapes of the new molecules strongly depend on the characteristics of the heptad repeat patterns. The highest similarities between parent and retro-proteins are associated with the presence of uninterrupted heptad patterns in helical bundles sequences.

Synthesis and Physicochemical Characterization of Undecylenic Acid Grafted to Hyaluronan for Encapsulation of Antioxidants and Chemical Crosslinking.

In this work, a new amphiphilic derivative made of 10-undecylenic acid grafted to hyaluronan was prepared by mixed anhydrides. The reaction conditions were optimized, and the effect of the molecular weight (Mw), reaction time, and the molar ratio of reagents was explored. Using this methodology, a degree of substitution up to 50% can be obtained. The viscosity of the conjugate can be controlled by varying the substitution degree. The physicochemical characterization of the modified hyaluronan was performed by infrared spectroscopy, Nuclear Magnetic Resonance, Size-Exclusion Chromatography combined with Multiangle Laser Light Scattering (SEC-MALLS), and rheology. The low proton motility and self-aggregation of the amphiphilic conjugate produced overestimation of the degree of substitution. Thus, a novel method using proton NMR was developed. Encapsulation of model hydrophobic guest molecules, coenzyme Q10, curcumin, and α-tocopherol into the micellar core was also investigated by solvent evaporation. HA-UDA amphiphiles were also shown to self-assemble into spherical nanostructures (about 300 nm) in water as established by dynamic light scattering. Furthermore, HA-UDA was crosslinked via radical polymerization mediated by ammonium persulphate (APS/TEMED). The cross-linking was also tested by photo-polymerization catalyzed by Irgacure 2959. The presence of the hydrophobic moiety decreases the swelling degree of the prepared hydrogels compared to methacrylated-HA. Here, we report a novel hybrid hyaluronan (HA) hydrogel system of physically encapsulated active compounds and chemical crosslinking for potential applications in drug delivery.

Synthesis and structure characterization of sulfated galactomannan from fenugreek gum

Sulfated derivatives of the galactomannan family have multiple biological activities. In the present study, sulfated galactomannan from fenugreek gum (SFG) were prepared by chlorosulfuric acid/pyridine (CSA/Py) method. In order to acquire the derivatives with the highest degree of substitution (DS), the experiment optimum conditions of the sulfation were determined by Box-Behnken design (BBD). The quadratic regression model analysis confirmed that reaction time was the most significant parameter for the impact of DS. Under the selected conditions, maximum DS was obtained as 0.490. Results of FT-IR and X-ray photoelectron spectroscopy (XPS) indicated the present of SO3− group. In 13C NMR spectroscopy, the original C-6 peaks did not disappear completely and the newly peaks appeared at δ 63.2 and 64.0 illustrating the incomplete substitution were predominantly on C-6 position. After sulfation, size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS) detected all the weight average molecular mass (MW) of sulfated derivatives decrease rapidly. The introduction of the electrostatic interaction of negatively charged SO3− groups and the decrease in MW could have a significant effect on its biological activity.

Synthesis and structural features of phosphorylated Artemisia sphaerocephala polysaccharide

Phosphorylation is a key route to achieve varieties of biological activities for polysaccharides. Currently, conventional phosphorylation reagents are characterized by long processing times and high reaction temperatures. Here, we reported phosphorylation of Artemisia sphaerocephala polysaccharides (PASP) with the degrees of substitution (DS) of 0.34-0.54 in mild reaction conditions (3–4h and 25°C) by employing POCl3/pyridine technique. FT-IR spectroscopy and X-ray photoelectron spectroscopy (XPS) analysis confirmed the appearance of phosphate ester (P5+) groups in PASP. Size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS) result clearly showed temperature-dependent increment of weight average molecular mass (MW). High temperature facilitated the reaction of phosphoryl chloride with primary and secondary hydroxyl groups, favoring the crosslinking of polysaccharide chains by phosphate di-esters as further confirmed in 13C NMR determination. Our results provide insight into the relationship between the structure and reaction conditions of phosphorylated polysaccharide, which is important for understanding and exploiting these derivatives in a wide range of applications.

Microwave-assisted synthesis, structure and anti-tumor activity of selenized Artemisia sphaerocephala polysaccharide

In this work, selenylation of Artemisia sphaerocephala polysaccharide (SeASPMW) was studied by using H2SeO3/HNO3/BaCl2 reaction system in microwave field. SeASPMW exhibited the Se content range of 111–264μg/g with high yields (72.1–94.9%). 13C NMR results indicated that the weak C-6 substitution was occurred. The decrease (from 7.348×104g/mol to 1.736–4.667×104g/mol) in weight average molecular mass (MW) of SeASPMW was observed in size exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS) analysis. SeASPMW exhibited a more rigid solution conformation which might be due to the degradation of polysaccharide chains in acidic reaction reagent. This was also supported by atomic force microscopy (AFM) result that SeASPMW showed short chains and island-like topography. In anti-tumor activity assays, SeASPMW6 exhibited the inhibition rates of 32.381% and 39.776% against human non-small cell lung cancer cell line (H1650) at the concentration of 100 and 200μg/mL, respectively. The relatively weak inhibition effect of SeASPMW was not related to cell apoptosis and cell cycle arrest, suggesting Se content might be a key factor to influence the anti-tumor activities of selenized polysaccharides in vitro.

Synthesis of selenium-containing Artemisia sphaerocephala polysaccharides: Solution conformation and anti-tumor activities in vitro

It has been reported in our previous work that selenized Artemisia sphaerocephala polysaccharides (SeASPs) with the Se content range of 168–1703μg/g were synthesized by using Na2SeO3/HNO3/BaCl2 system. In the present work, the solution property of SeASP was studied by using size exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS). A decrease in df values indicated that SeASPs with different conformational features that were highly dependent on MW. SeASPs exhibited a more rigid conformation (df value of 1.29–1.52) in low molecular weight range (MW of 1.026–1.426×104g/mol) and compact spherical conformation in high molecular weight range (MW of 2.268–4.363×104g/mol). It could be due to the degradation of polysaccharide chains in HNO3, which was supported in monosaccharide composition analysis. Congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results also confirmed the conformational transition and the evidence on the shape of the rigid chains. In vitro anti-tumor assays, SeASP2 displayed greater anti-proliferative effects against three tumor cell lines (hepatocellular carcinoma HepG-2 cells, lung adenocarcinom A549 cells and cervical squamous carcinoma Hela cells) in a dose-dependent manner. This suggested that selenylation could significantly enhance the anti-tumor activities of polysaccharide derivatives in vitro.

Regioselective sulfation of Artemisia sphaerocephala polysaccharide: Solution conformation and antioxidant activities in vitro

Regioselective modification is an effective approach to synthesize polysaccharides with different structure features and improved properties. In this study, regioselective sulfation of Artemisia sphaerocephala polysaccharide (SRSASP) was prepared by using triphenylchloromethane (TrCl) as protecting precursor. The decrease in fractal dimension (df) values (1.56–2.04) of SRSASP was observed in size-exclusion chromatography combined with multi angle laser light scattering (SEC-MALLS) analysis. Compared to sample substituted at C-6, SRSASP showed a more expanded conformation of random coil, which was attributed to the breakup of hydrogen bonds and elastic contributions. Circular dichroism (CD), methylene blue (MB) and congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results confirmed the conformational transition and stiffness of the chains after sulfation. SRSASP exhibited enhanced antioxidant activities in the DPPH, superoxide and hydroxyl radical scavenging assay. Sulfation at C-2 or C-3 was favorable for the chelation which might prevent the generation of hydroxyl radicals. It concluded that the degree of substitution and substitution position were the factors influencing biological activities of sulfated polysaccharides.

Molecular characteristics of an exopolysaccharide from Lactobacillus rhamnosus KF5 in solution

The molecular weight and chain conformation of an exopolysaccharide (EPS), S2, from Lactobacillus rhamnosus KF5 were determined by size-exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), viscometry and atomic force microscopy (AFM). The weight-average molecular weight (Mw), intrinsic viscosity [ƞ], radii of gyration (Rg) and hydrodynamic radii (Rh) were 7.5×105Da, 2.331dL/g, 44.3nm and 29.4nm, respectively. The conformational parameters were calculated from the above data according to the theory of dilute polymer solutions. The Mark–Houwink–Sakurada exponent α was found to be 0.687, and the value of the structure-sensitive parameter ρ (Rg/Rh) was 1.51. The results revealed that the EPS S2 existed as a random coil conformation in 0.1M NaNO3 aqueous solution. AFM further confirmed the random coil morphology of this molecule in aqueous solution. EPS S2 present extended chains (fibrous morphology), with circular side chains, dispersed in SDS solution.

Catalytic synthesis of sulfated polysaccharides. II: Comparative studies of solution conformation and antioxidant activities

Sulfated derivatives of Artemisia sphaerocephala polysaccharide (ASP) with high degree of substitution (DS) were synthesized using 4-dimethylaminopyridine (DMAP)/dimethylcyclohexylcarbodiimide (DCC) as catalyst. Size exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS) results showed a decrease in fractal dimension (df) values of sulfated ASP (SASP). Compared to ASP and SASP with low DS (0.51–1.01), SASPcata2 exhibited an internal structure between rigid rod and random coil with a DS of 1.24. DS had greater influence on its conformation in aqueous solution. Circular dichroism (CD), methylene blue (MB) and Congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results confirmed that the degradation of ASP and SO3H groups improved significantly the stiffness of the chains due to the electrostatic effect. Furthermore, antioxidant experiments revealed that high DS could enhance the scavenging activities of radicals and reducing power of SASP in vitro. The extended chain conformation was beneficial to enhance the biological activity of sulfated polysaccharides.

Optimization of the solid-state fermentation and properties of a polysaccharide from Paecilomyces cicadae (Miquel) Samson and its antioxidant activities in vitro.

The culture conditions for the yield of a polysaccharide (PCPS) produced by Paecilomyces cicadae (Miquel) Samson on solid-state fermentation were investigated using response surface methodology (RSM). Plackett–Burman design (PBD) was applied to screen out significant factors, followed by the paths of steepest ascent to move to the nearest region of maximum response. Then Box-Behnken design (BBD) was conducted to optimize the final levels of the culture conditions. After analyzing the regression equation and the response surface contour plots, relative humidity 56.07%, inoculum 13.51 mL/100 g and temperature 27.09°C were found to be the optimal key parameters for PCPS production. The maximum predicted yield of PCPS was 10.76 mg/g under the optimized conditions. The resulting PCPS (FPCPS) generated at optimal conditions was purified by chromatography column and found to be composed of mannose (43.2%), rhamnose (32.1%), xylose (14.5%) and arabinose (10.2%). Based on the size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS) analysis, FPCPS adopted a Gaussian coil conformation in 0.1 M NaNO3 solution with 3.75×106 g/mol of the weight-average molar mass (Mw) and 41.1 nm of the root-mean square radius (Rg2)z 1/2. Furthermore, both of the polysaccharides were revealed to have strong antioxidant activities by evaluating in DPPH radical, superoxide radicals and hydroxyl radical assay. These data suggest the polysaccharides of Paecilomyces cicadae (Miquel) Samson produced by solid-state fermentation could be explored as potential natural antioxidants.

SEC-MALLS analysis of TEMPO-oxidized celluloses using methylation of carboxyl groups

Two cellouronic acids [sodium (1 → 4)-β-polyglucuronates, CUAs] and one 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized wood cellulose (TOC) became soluble in 8 % lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) after the methylation of C6 carboxyl groups in these samples using trimethylsilyldiazomethane (TMSD). The obtained solutions were diluted to 1 % LiCl/DMAc and subjected to size-exclusion chromatography combined with multi-angle laser-light scattering (SEC-MALLS). Neither depolymerization nor side reactions took place during methylation; this was confirmed by SEC-MALLS and nuclear magnetic resonance analyses, using CUAs as models. The SEC-MALLS analysis of the original wood cellulose and the carboxyl-methylated TOC prepared from it, using 1 % LiCl/N,N-dimethyl-2-imidazolidinone and 1 % LiCl/DMAc, respectively, as eluents, showed that the weight-average degree of polymerization of the original wood cellulose decreased from 3,100 to 2,210 through TEMPO-mediated oxidation. The molecular-mass distributions of the original wood cellulose and the TOC both consisted of one large peak with a small shoulder, indicating that some of the oxidized hemicelluloses remained in the TOC. The combination of methylation of carboxyl groups in polysaccharides using TMSD and subsequent SEC-MALLS analysis using 1 % LiCl/DMAc as an eluent may be applicable not only to TOCs, but also to other polysaccharides with carboxyl groups, for evaluation of their molecular-mass parameters.

Effect of Heating on Chain Conformation of Branched β-Glucan in Water

The thermal stability of polysaccharides under heat treatment is an important factor to their functionality in food and pharmaceutical fields. The stiff branched β-glucan coded as AF1-1 isolated from Auricularia auricula-judae was investigated with viscometry, dynamic light scattering (DLS), and size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) in water at 25 to 170 °C. The chain conformation of AF1-1 in the aqueous solution exhibited a sharp decrease in viscosity, hydrodynamic radius (Rh), and weight-average molecular weight (Mw) at elevated temperature in a narrow range of 140 to 160 °C. It was confirmed that the conformation transitions of the AF1-1 chains from rod-like chains to the flexible occurred during heating to 140-160 °C for 30 min, leading to the coexistence of the flexible chains and stiff chains at 155 °C as a result of the breaking of the intra- and intermolecular hydrogen bonds of the AF1-1 macromolecules. The results from scanning electron microscopy and atomic force microscopy further directly proved that the AF1-1 nanofibers in water were destructed into flexible coils consisting of individual chain at the elevated temperature higher than 155 °C, supporting the conformation transition. The conformational transition from stiff to flexible chains at 140-160 °C was irreversible. However, the chain shape and stiffness of AF1-1 was stable below 140 °C and hardly changed with an increase in the temperature. This was important for the application in the fields of food and pharmaceutical.

Effects of alkali dissociation on the molecular conformation and immunomodulatory activity of longan pulp polysaccharide (LPI)

The effects of alkali dissociation on the molecular conformation and immunomodulatory activity of longan pulp polysaccharide (LPI) were investigated to explore their possible relationship. The molecular conformations of LPI and its degraded derivatives (LPI1 and LPI2) were examined by size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS), Congo red test and atomic force microscopy (AFM). The results confirmed the transformation of LPI from compact sphere-like conformation to slightly dissociated sphere-like conformation (LPI1) or single-helix chain (LPI2). Compared with the control, splenocyte proliferation and NK cell cytotoxicity were significantly enhanced by LPI1 and LPI2 (P 0.05). All the polysaccharides could significantly enhance macrophage phagocytosis at 100 or 200 μg/mL (P < 0.05). Single-helix chain might play an important role in activating lymphocytes and NK cells, but having weak contribution to macrophage phagocytosis.

Thermally Induced Conformation Transition of Triple-Helical Lentinan in NaCl Aqueous Solution

Lentinan, a beta-(1-->3)-D-glucan, was isolated from Lentinus edodes by using an improved extraction and purification method to show good water solubility and high yield. The results from 13C NMR, size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), and optical rotation revealed that lentinan existed in a triple-helical conformation in the aqueous solution at 25 degrees C, whereas the thermally induced conformation transition from triple helix to single flexible chains occurred at elevated temperatures. The dependences of the weight-average molecular weight (Mw), radius of gyration (<s2>z1/2), hydrodynamic radius (Rh), intrinsic viscosity ([eta]), and specific optical rotation of lentinan on temperature in 0.9% NaCl aqueous solution showed an abrupt drop at 130-145 degrees C. It was confirmed that the conformation transitions from triple strand to single chain and from extended chains to winding chains for lentinan were completed rapidly at 130-145 degrees C, as a result of the simultaneous destruction of the intra- and intermolecular hydrogen bonds in lentinan. The thermally induced conformational transition was irreversible. The results from atomic force microscopy (AFM) and DLS demonstrated the existence of intrachain entanglement for the triple-helical chains, leading to the wormlike linear, circular, and crossover species for lentinan having high Mw (1.71x10(6)) in aqueous solution at 25 degrees C.

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

A (1→3)-β- 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 (< S 2> z 1/2), and intrinsic viscosity ([ η]) of lentinan and its fractions in 0.9% NaCl aqueous solution and dimethyl sulfoxide (Me 2SO) were determined by size-exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS), LLS, and viscometry. Analysis of M w, [ η], and < S 2> 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 [ α] D of lentinan in water–Me 2SO 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 2SO. 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 × 10 6 exhibited the highest antitumor activity in vivo, having an inhibition ratio ( ξ) of 49.5%, close to that of 5-fluorouracil ( ξ = 50.5%), whereas the bioactivity ( ξ = 12.3%) of its single flexible chains almost disappeared. The triple-helix conformation plays an important role in enhancing the antitumor effects of lentinan.

Solution properties of water-insoluble polysaccharides from the mycelium of Ganoderma tsugae

Two kinds of water-insoluble polysaccharides (GM5-1 and GM6-1) were isolated from the mycelium of Ganoderma tsugae with 0.5 M NaOH aqueous solution at 25 and 65 °C successively. Their chemical structures were characterized by infrared spectroscopy, gas chromatography and 13C NMR. GM5-1 was composed of (1→3)-α- d-glucan and mannoxylan, while GM6-1 was mainly mannoxylan. The sample GM6-1 was fractionated into five fractions by nonsolvent addition, and their weight-average molecular mass ( M w) and intrinsic viscosity [ η] were determined by laser light scattering (LLS), size-exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS) and viscometer in 0.25 M LiCl/DMSO at 30 °C. The M w dependences of [ η] and the radius of gyration ( 〈 S 2 〉 z 1 / 2 ) were found to be [ η]=8.9×10 −2 M w 0.57 (cm 3 g −1) and 〈 S 2 〉 z 1 / 2 =4.1×10 −2 M w 0.52 (nm) for GM6-1 in the M w range from 1.50×10 6 to 4.81×10 6, implying a random coil conformations in the solution. Molecular parameters of GM6-1 were calculated on the basis of the wormlike cylinder chain to be 4.2 nm for persistent length ( q) and 832 nm −1 for molar mass per unit contour length ( M L).

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 <s(2)>(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 <s(2)>(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.

Specific degradation of hyaluronic acid and its rheological properties

High molecular hyaluronic acid was degraded by ultrasonication to definite molecular weight varying from 0.4 to 2.0 × 10 6g mol −1. The weight average molecular weight (M w) of degraded hyaluronic acid in 0.2 M NaCl was determined by size exclusion chromatography combined with multi-angle-laser-light-scattering (SEC-MALLS). Comparison measurements by using an Ubbelohde viscosimeter and a low shear rate viscosimeter (Zimm-Crothers) were performed for all sonicated samples. The relation of M w of the hyaluronic acid to the intrinsic viscosity [η] was calculated according to the Mark-Houwink equation, [η] = K M w α. The coefficients K = 5.075 10 −2 and α = 0.716 determined by Ubbelohde and K = 3.023 10 −2 and α = 0.770 by Zimm-Crothers viscosimeter. Furthermore, the dependence on pH and the influence of bivalent cations were investigated. In the range of pH 5.0 to 11.0 the intrinsic viscosity [η] was constant. At pH less than 5.0, the intrinsic viscosity [η] and the molecule volume decreased probably due to the protonation of glucoronic acid groups. Most bivalent cations do not affect the rheological behaviour significantly, only Fe 2+ causes to decrease of the intrinsic viscosity [η] and the molecule volume by intramolecular chain linkage combined with displacement of hydrating water.