Beta-galactosidase (Escherichia coli) has a second catalytically important Mg2+ site

It is shown here that Escherichia coli beta-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the k(cat) (with oNPG as the substrate) to increase about 100 s(-1); the Km was not affected. The Kd for binding the second Mg2+ is about 10(-4)M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k(cat) increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.     

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].     

Crystal structure of truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.     

Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.     

Hen egg white lysozyme as an inhibitor of mushroom tyrosinase

We report a kinetics study on hen egg white lysozyme's (HEWL) inhibitory effect on mushroom tyrosinase catalysis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) or L-tyrosine. For the first time, we demonstrate HEWL as a robust inhibitor against mushroom tyrosinase in catalysis of both substrates. The kinetics pattern matches a mixed (mostly non-competitive) partial inhibition. Ki and ID50 value of HEWL are more than 20-fold lower than that of kojic acid, a well-known chemical inhibitor of mushroom tyrosinase. Ki, alpha value and beta value, are almost identical in both experiments (L-DOPA and L-tyrosine as substrates, respectively), which suggests this common inhibition mechanism affects both steps. The inhibitory effect increases as both proteins were mixed and pre-incubated for less than 1 h. HEWL-depletion only removed about half of the inhibitory effect. Here we propose a novel function of HEWL, which combines the reversible inhibition and the irreversible inactivation toward mushroom tyrosinase. Discovery of HEWL as an inhibitor to mushroom tyrosinase catalysis may be commercially valuable in the food, medical and cosmetic industries.     

Oxidation of L-serine and L-threonine by bis(hydrogen periodato)argentate(III) complex anion: a mechanistic study

Oxidation of l-serine and l-threonine by a silver(III) complex anion, [Ag(HIO(6))(2)](5-), has been studied in aqueous alkaline medium. The oxidation products of the amino acids have been identified as ammonia, glyoxylic acid and aldehyde (formaldehyde for serine and acetaldehyde for threonine). Kinetics of the oxidation reactions has been followed by the conventional spectrophotometry in the temperature range of 20.0-35.0 degrees C and the reactions display an overall second-order behavior: first-order with respect to both Ag(III) and the amino acids. Analysis of influences of [OH(-)] and [periodate] on the second-order rate constants k' reveals an empirical rate expression: k(')=(k(a)+k(b)[OH(-)])K(1)/([H(2)IO(6)(3-)](e)+K(1)), where [H(2)IO(6)(3-)](e) is equilibrium concentration of periodate, and where k(a)=6.1+/-0.5M(-1)s(-1), k(b)=264+/-6M(-2)s(-1), and K(1)=(6.5+/-1.3)x10(-4)M for serine and k(a)=12.6+/-1.7M(-1)s(-1), k(b)=(5.5+/-0.2)x10(2)M(-2)s(-1), and K(1)=(6.2+/-1.5)x10(-4)M for threonine at 25.0 degrees C and ionic strength of 0.30M. Activation parameters associated with k(a) and k(b) have also been derived. A reaction mechanism is proposed to involve two pre-equilibria, leading to formation of an Ag(III)-periodato-amino acid ternary complex. The ternary complex undergoes a two-electron transfer from the coordinated amino acid to the metal center via two parallel pathways: one pathway is spontaneous and the other is assisted by a hydroxide ion. Potential applications of the Ag(III) complex as a reagent for modifications of peptides and proteins are implicated.     

Characterization of a GDP-D-mannose 3',5'-epimerase from rice

The enzymatic characterization of GDP-d-mannose 3',5'-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.     

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.     

Secondary metabolites from Opuntia ficus-indica var. saboten

A butanol fraction, from the methanolic extract of Opuntia ficus-indica var. saboten, on purification either by preparative TLC or reversed phase HPLC, yielded three chemical components: isorhamnetin 3-O-(6'-O-E-feruloyl)neohesperidoside (1), (6R)-9,10-dihydroxy-4,7-megastigmadien-3-one-9-O-beta-D-glucopyranoside (2) and (6S)-9,10-dihydroxy-4,7-megastigmadien-3-one-9-O-beta-D-glucopyranoside (3) along with 15 known compounds. Structures of compounds (1-3) were elucidated by aid of spectroscopic analyses. The absolute stereochemistry in compounds 2 and 3 was established with the help of CD data analysis and comparison with the literature data. In a DPPH radical scavenging assay, compound 1 showed moderate inhibitory activity (IC50 = 45.58 microg/ml).     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Asymmetric synthesis of methyl 6-deoxy-3-O-methyl-alpha-L-mannopyranoside from a non-carbohydrate precursor

A novel method is reported for preparing methyl 6-deoxy-3-O-methyl-alpha-L-mannopyranoside (1) by asymmetric synthesis, using 2-acetylfuran (2), a non-chiral simple molecule, as the starting material and achieving high yields via (S)-1-(2-furyl)ethanol and (S)-1-(2,5-dihydro-2,5-dimethoxy-2-furyl)ethanol.     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

The structure of the carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-79104

The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: [carbohydrate structure: see text] where R is H or 4-O-Me-alpha-L-Fuc-(1-2)-4-O-Me-beta-Hep-(1-3)-alpha-GlcNAc-(1-2)-beta-Man-(1-3)-beta-ManNAc-(1-4)-alpha-Gal-(1-4)-beta-Hep-(1-3)-beta-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Sugar binding in lactose permease: anomeric state of a disaccharide influences binding structure

Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with β(α)-d-galactopyranosyl-(1,1)-β-d-galactopyranoside (ββ-(Galp)₂) and αβ-(Galp)₂ result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and αβ-(Galp)₂ has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp)₂ than ββ-(Galp)₂, i.e. −10.7(±0.7) and −3.1(±1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with ββ-(Galp)₂ binding resulting in larger distortion than αβ-(Galp)₂.     

The inhibitory effects of flavonoids and antiestrogens on the Glut1 glucose transporter in human erythrocytes

Flavonoids and isoflavonoids are potent inhibitors of glucose efflux in human erythrocytes. Net changes of sugars inside the cells were measured by right angle light scattering. The inhibitory potency of hydroxylated flavonoids depends on the pH of the medium. The apparent affinity is maximal at low pH where the molecule is in the undissociated form. The following K(i)-values at pH 6.5 in microM have been obtained: phloretin 0.37+/-0.03, myricetin 0.76+/-0.42, quercetin 0.93+/-0.28, kaempferol 1.33+/-0.17, isoliquiritigenin 1.96, genistein 3.92+/-0.62, naringenin 8.88+/-1.88, 7-hydroxyflavone 17.58+/-3.15 and daidzein 18.62+/-2.85. Flavonoids carrying hydroxyl groups are weak acids and are deprotonated at high pH-values. From spectral changes pK-values between 6.80 (naringenin) and 7.73 (myricetin) have been calculated. No such pK-value could be obtained from quercetin which was rather unstable at alkaline pH. Flavone itself without a hydroxyl group does not demonstrate any absorbance changes at different pH-values and no significant change in inhibition of glucose transport with pH (K(i)-value around 35 microM). In this respect it is similar to the antiestrogens diethylstilbestrol, tamoxifen and cyclofenil with K(i)-values for glucose efflux inhibition of 2.61+/-0.30, 6.75+/-2.03 and 3.97+/-0.54 microM. Except for phloretin, the flavonoids investigated have planar structures. The inhibitory activity in glucose efflux of planar flavonoids increases exponentially with the number of hydroxyl groups in the molecule.     

Investigation of the functional relevance of the catalytically important Glu(28) in family 51 arabinosidases

The alpha-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus is a family 51 glycosyl hydrolase. According to classification hierarchy, family 51 belongs to clan GH-A. While the major GH-A motifs, the catalytic acid-base and nucleophile, are conserved in AbfD3, a third catalytically important residue (Glu(28)) does not appear to be analogous to any known GH-A motif. To evaluate the importance of Glu(28), bioinformatics analyses and site-saturation mutagenesis were performed. The results indicate that Glu(28) forms part of a family 51 arabinosidase motif which might be functionally homologous to a conserved N-terminal motif found in exo-acting enzymes from families 1 and 5. Importantly, the data reveal that Glu(28) is a key determinant of substrate recognition in the -1 subsite, where it may also play an important role in water-mediated deglycosylation of the glycosyl-enzyme covalent intermediate.     

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.     

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.