Structural characterisation of the olive pomace pectic polysaccharide arabinan side chains

An arabinan (97% of Ara and 3% of hexuronic acid) was isolated from the alcohol-insoluble residue (AIR) of olive pomace by treatment with 0.02 M HNO(3), at 80 degrees C, followed by graded precipitation with ethanol. It was separated from acidic pectic polysaccharides by anion-exchange chromatography, and by size-exclusion chromatography its molecular weight was estimated as 8.4 kDa. By methylation analysis, the linkage composition was established as 5:4:3:1 for (1-->5)-Araf, T-Araf, (1-->3,5)-Araf and (1-->3)-Araf, respectively. 13C NMR spectroscopy confirmed this linkage composition, and allowed to assign the alpha anomeric configuration for the arabinofuranosyl residues, except for some terminally linked ones, that were seen to occur as T-beta-Araf. By 2D NMR spectroscopy (1H and 13C), it was possible to conclude that the T-beta-Araf was (1-->5)-linked to a (1-->5)-Araf residue. Also, in the arabinan (1-->5)-Araf backbone, the branched (1-->3,5)-Araf residues were always adjacent to linear (1-->5)-Araf residues. A tentative structure is proposed.     

Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica

Abstract The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans ( S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).     

Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels

Amylomaltases are 4-alpha-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer alpha-1,4-linked glucans to another acceptor, which can be the 4-OH group of an alpha-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95 degrees C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase.     

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.     

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G _i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K _m and higher k _cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.     

Discontinuous volume transitions induced by calcium–sodium ion exchange in anionic gels and their neurobiological implications

Discontinuous volume transitions in poly(acrylic acid) gels were studied by determining equilibrium swelling curves of small gel beads immersed in salt solutions containing varying concentrations of Ca²⁺ and Na⁺. Reversible contractions of gel membranes associated with Ca²⁺-Na⁺ exchange were examined using both isometric and isotonic recording devices. A discontinuous change in electric conductance was demonstrated in association with Ca²⁺-Na⁺ exchange in gel membranes. These experimental findings provide a sound physicochemical basis for elucidating the mechanism of nerve excitation. © 1994 John Wiley & Sons, Inc.     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Structural analysis of a novel anionic polysaccharide from Porphyromonas gingivalis strain W50 related to Arg-gingipain glycans

The Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cell-surface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of alpha-1,6-linked mannose residues and the side-chains contain alpha-1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Manalpha1-2Manalpha1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O-phosphorylation of APS abolished cross-reactivity suggesting that Manalpha1-2Manalpha1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.     

Structural properties of colloidal complexes between condensed tannins and polysaccharide hyaluronan

Interactions of plant tannins with polysaccharide hyaluronan are studied by means of light scattering and small-angle X-ray scattering (SAXS). In this paper, we show that (1) the tannin-polysaccharide complexes remain stable in colloidal suspension; (2) the masses and structures of colloidal tannin-polysaccharide objects depend on the tannin degree of polymerization; and (3) the densities of tannin-polysaccharide aggregates are about 7 times lower than the density of a single solvated polysaccharide molecule. Short tannins and polysaccharides are aggregated in loose oligomeric structures whose sizes are comparable to a single polysaccharide molecule. Tannins longer than 10 nm and polysaccharides are aggregated in larger microgel-like particles whose sizes exceed 200 nm.     

Water dynamics in charged and uncharged polysaccharide gels by quasi-elastic neutron scattering

Using a microeV neutron spectrometer we have studied the mobility of water in gels formed by two polysaccharides: agarose and hyaluronic acid. Agarose is a nearly uncharged polysaccharide; its gels are fairly stiff, quasi-random networks of fibre bundles. Hyaluronic acid is a highly charged polysaccharide capable of retaining large amounts of water in entangled meshworks with unusual rheological properties. We have analysed sets of quasi-elastic lineshapes broadened by two proton populations with different degrees of freedom. The resulting microscopic mobility parameters and their temperature dependence reveal a complex behaviour. The overall effect of the biopolymer network is to increase translational as well as rotational relaxation times, but the changes observed are not dramatic and cannot fully account for the strikingly different macroscopic properties of these gels. Local electrostatic interactions (over 3 to 20 A) do not appear to influence significantly the rheological behaviour.     

Chiroptical characterisation of polysaccharide secondary structures in the presence of interfering chromophores: Chain conformation of inter-junction sequences in calcium alginate gels

The changes in chain conformation which accompany Ca²⁺-induced gelation of alginate have been investigated by a combined circular dichroism (c.d.) and optical rotatory dispersion (o.r.d.) approach. C.d. changes in the carboxyl n→π^* spectral region, arising predominantly from formation of calcium poly-l-guluronate junctions, were monitored for three alginates of widely differing block composition. The corresponding o.r.d. changes, calculated by Kronig-Kramers trnasform, were subtracted from the observed changes in o.r.d. on gelation, to “unmask” the changes in optical activity of the conformation-sensitive electronic transitions of the polysaccharide backbone. Contributions to the “residual” o.r.d. difference spectra from poly-l-guluronate, poly-d-mannuronate, and heteropolymeric chain-sequences were calculated by solution of simultaneous equations at each wavelength. Results for poly-guluronate sequences are in agreement with previous studies of alginate films by vacuum ultraviolet c.d., and with observed c.d. and o.r.d. changes on addition of calcium ions to homopolyguluronate segments in solution. The much greater changes in backbone optical activity calculated for polymannuronate and heteropolymeric chain-sequences, however, have no counterpart in the behaviour of these sequences in isolation. An explanation is proposed in terms of stretching of interconnecting sequences between calcium polyguluronate junctions in alginate gels, to give a more-extended chain conformation than in free solution.     

Hemoglobin-dialdehyde dextran conjugates: Improvement of their oxygen-binding properties with anionic groups

We studied the conjugates formed between hemoglobin and sulfated or unsulfated oxidized dextran. It appears that the presence of sulfated groups favors imino bond formation between the protein and the polymer, as the average molecular size of the conjugates is larger in this case. Under neutral conditions, the oxygen-binding properties of the conjugates depend on the presence or absence of oxygen during the coupling reaction. With unsulfated dextran, oxyhemoglobin leads to conjugates with increased oxygen affinity (P ₅₀/P ₅₀ native hemoglobin 0.5) compared to that of free hemoglobin (P ₅₀=4 mm Hg), whereas deoxyhemoglobin leads to conjugates with decreased oxygen affinity (P ₅₀/P ₅₀ native hemoglobin 3). The use of sulfated dextran reinforces this lowering in oxygen affinity, which indicates that sulfated dextran acts as a permanent macromolecular effector of hemoglobin (P ₅₀/P ₅₀ native hemoglobin 4). Moreover, it can be assumed that some of the linkages involve the 2,3-diphosphoglycerate binding site, as the strong effector inositol hexaphosphate has only a slight effect on the oxygen-binding properties of the conjugate prepared in the deoxy state (P ₅₀/P ₅₀ native hemoglobin close to 4.4 and 6, respectively, for unsulfated and sulfated conjugates). Although dextran substituted with benzenehexacarboxylic acid (BHC) leads to a low-oxygen-affinity conjugate when linked to oxyhemoglobin through amide bonds (P ₅₀/P ₅₀ native hemoglobin 5), oxidized dextran modified with BHC leads, with oxyhemoglobin, to a conjugate whose oxygen affinity is close to that of free hemoglobin (P ₅₀/P ₅₀ native hemoglobin 1.2).     

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC–MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.     

Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.     

Arabinoxylan gels: impact of the feruloylation degree on their structure and properties

Arabinoxylan (AX) samples of decreasing ferulic acid (FA) contents were chemically prepared from water-extractable wheat arabinoxylans without affecting their other structural properties. Gels were obtained from these partially feruloylated WEAX (PF-WEAX) by enzymatic covalent cross-linking of FA leading to the formation of diferulic (di-FA) and tri-ferulic acid (tri-FA). WEAX gelling ability was found related to the WEAX FA content whereas the gel structure and properties depended on the density of newly formed covalent cross-links. FA content of WEAX ranging from 1.4 to 2.3 microg/mg AX gave gels with di-FA cross-links contents from 0.20 to 0.43 microg/mg AX and G' values from 5 to 44 Pa. For WEAX gels with initial FA contents from 1.6 to 2.3 microg/mg AX, average mesh size ranging from 331 to 263 nm were calculated from swelling experiments. Cross-linking densities of gels, determined from swelling experiments, were higher than those that could be theoretically estimated from the di-FA and tri-FA content of WEAX gels. This result suggests that, in addition to di-FA and tri-FA, higher ferulate cross-linking and physical entanglements would contribute to the final WEAX gel structure.     

Structural properties of pectin-gelatin gels. Part II: effect of sucrose/glucose syrup

Small deformation dynamic oscillation and bright field microscopy were used to examine the structural properties of single and mixed high methoxy pectin and gelatin systems in the presence of sucrose/glucose syrup blends. Co-solute concentrated (≥78%) systems of the polysaccharide form rubbery structures which are readily transformed into glassy consistencies according to the time-temperature superposition principle. Increasing amounts of co-solute in the gelatin samples induce changes in viscoelasticity from that of conventional hydrogels to mechanical traces that cover much of the plateau region and the beginning of the glass transition area. Furthermore, manipulation of the protein/ sugar ratio can result in strong crystalline matrices, or viscoelastic solutions where the co-solute forms the continuous phase and the gelatin inclusions can undertake a conformational transition. The properties of the single components were used to rationalise the phase behaviour of their mixtures. Upon triggering the gelation of pectin, mixtures can be made where either gelatin or both components form a continuous phase. Results are discussed in the light of evidence obtained from the ethylene glycol work in Part I.     

Structural properties of signal peptides and their membrane insertion

Structural properties of the amino acid sequences from 22 signal peptides have been analyzed and compared with peptides known to interact with biological membranes and liposomes, melittin, a lytic peptide of bee venom, and the non-polar C-terminal segment of cytochrome b5. All these peptides evidence a double amphipatic structure with an hydrophobic core of 9 to 24 amino acid residues and two charged polar ends. They all exhibit a high potential for making alpha-helix and, to a lesser degree, extended or beta-sheet conformation with low or negative potentials for making reverse turns or aperiodic conformation. A model of spontaneous insertion of these peptides into the lipid bilayer without specific surface receptor protein is proposed, where the two polar ends interact with each polar face of the lipid bilayer and the hydrophobic core inserts into the non-hydrogen bonding environment of the fatty acid side chains. This insertion could be the molecular trigger for ribophorin assembly around the signal peptide and subsequent attachment to the ribosome prior to the transfer of the polypeptide chain through the endoplasmic reticulum membrane.     

Physical properties of polyethyleneimine-alginate gels

Summary The procedure for preparation of polyethyleneimine (PEI)-alginate gel and some of its physical properties are presented. The rigidity modulus (G) of the gel appears to increase to the second power of PEI concentration. Shrinkage is linearly related to pH up to pH 6.0. G increases as the pH is lowered, and decreases after freezing. The gels does not dissolve on heating and are strong, but fairly brittle. They are resistant to proteolytic enzymes.