Plant Polysaccharide Array for Studying Carbohydrate-Binding Proteins.

The specificity of the most plant carbohydrate-binding proteins (CBP), many of which are known only through bioinformatic analysis of the genome, has either not been studied at all or characterized to a limited extent. The task of deciphering the carbohydrate specificity of the proteins can be solved using glycoarrays composed of many tens or even hundreds of glycans immobilized on a glass surface. Plant carbohydrates are the most significant natural ligands for plant proteins; this work shows that plant polysaccharides without additional modification can be immobilized on the surface, bearing N-hydroxysuccinimide activated carboxyl groups. As a result, an array of 113 well-characterized polysaccharides isolated from various plant cell walls, 23 mono- and oligosaccharides - components of polysaccharides, and glycans - ligands for widely known plant lectins was designed. Upon chemical immobilization of polysaccharides, their functional activity was preserved, which was confirmed by the results of interaction with antibodies and the plant lectin ricin. Using the constructed array, a previously unknown ability of ricin to bind polysaccharides was found, which significantly expands the knowledge of its specificity, and it was also found that a large variety of antibodies to plant polysaccharides are present in human peripheral blood.

Pectin from leaves of birch (Betula pendula Roth.): Results of NMR experiments and hypothesis of the RG-I structure.

We report that in birch leaf pectin, rhamnogalacturonan-I (RG-I) and galacturonan (HG) were found as separate polymers rather than domains of a complex macromolecule. RG-I and HG were separated by anion-exchange and size-exclusion chromatography and studied by using NMR spectroscopy. NMR spectra showed that methyl-esterified D-galactosyluronic acid residues were located only in HG. Oligosaccharides of similar structure to the backbone, but without terminal reducing residues in the NMR spectra, were found in RG-I. We hypothesize, these oligosaccharides and RG-I backbone can be covalently bound due to its co-eluted of from DEAE-cellulose and Sepharose CL-4B. This result differs from the classical RG-I model, which assumes that all Rhap and GalpA residues are located only in the RG-I backbone. In the heteronuclear multiple bond correlation (HMBC) and rotating frame Overhauser effect spectroscopy (ROESY) spectra, the correlation peaks confirming the substitution of 2,4-rhamnose residues at O-4 by only single D-galactose residues were identified.

Pectin-silica gels as matrices for controlled drug release in gastrointestinal tract.

The synthesis of pectin-silica gels for controlled drug release in gastrointestinal tract (GIT) using low methoxyl (LM) and high methoxy (HM) pectins and tetraethoxysilane (TEOS) as precursor is described. The FTIR spectra of the pectin-silica gels show intense absorption bands at 1246cm-1 and 802cm-1 corresponding to the vibrations COSi bonds, which absent in the FTIR spectra of the native pectins that indicate the formation covalent bond between silica and pectin macromolecules in the pectin-silica gels. Pectin-TEOS, pectin-Ca-TEOS and pectin-TEOS-Ca beads with mesalazine are synthesized by different combinations of sol-gel method using TEOS and ionotropic gelation method using calcium chloride. The best resistant of pectin-TEOS and pectin-Ca-TEOS beads during incubation in simulated gastric fluid for 2h and subsequently in simulated intestinal fluids for 18h is indicated. Pectin-TEOS beads are characterized by higher encapsulation efficiency (to 28%) than pectin-Ca-TEOS beads (to 16%). The drug release of pectin-silica beads in simulated GIT occurs gradually up to 80% and is directly dependent on the hardness of the beads. The surface morphology of beads is shown. The use of pectin-silica beads is promising with regard to the development of controlled release of drug formulations.

In vitro gastrointestinal-resistant pectin hydrogel particles for ß-glucuronidase adsorption.

Pectin hydrogel particles (PHPs) were prepared by ionotropic gelation of low methylesterified pectin of Tanacetum vulgare L. with calcium ions. Wet PHPs prepared from TVF exhibited a smaller diameter and the lower weight as well as exhibited the best textural properties in terms of hardness and elasticity compared to the PHPs prepared from commercial low methylesterified pectin (CU701) used for comparison. Upon air drying, PHPs prepared from CU701 became small and dense microspheres whereas the dry PHPs prepared from TVF exhibited a drop-like shape. The morphology of dry PHPs determined by scanning electron microscopy revealed that the surface of the TVF beads exhibited fibred structures, whereas the PHPs prepared from CU701 exhibited a smooth surface. The characterization of surface roughness using atomic force microscopy indicated less roughness profile of the PHPs prepared from TVF than CU701. PHPs prepared from TVF were found to possess in vitro resistance to successive incubations in simulated gastric (SGF), intestinal (SIF), and colonic fluid (SCF) at 37 °C for 2, 4 and 18 h, respectively. The PHPs prepared from CU701 swelled in SGF and then lost their spherical shape and were fully disintegrated after 4 h of incubation in SIF. The PHPs from TVF, which were subjected to treatment with SGF, SIF and SCF, were found to adsorb microbial ß-glucuronidase (ßG) in vitro. The data obtained offered the prospect for the development of the PHPs from TVF as sorbents of colonic ßG for the inhibition of re-absorption of estrogens.

Impact of cabbage pectin-protein complex on microbial ß-glucuronidase activity.

We previously demonstrated that pectin-protein complex (PPC) isolated from white cabbage adsorbs the ß-glucuronidase (ßG) enzyme of E. coli. Concurrently, we discovered a significant increase in ßG activity in the presence of PPC. The aim of this study is to identify the structural components of PPC that are responsible for ßG adsorption and activation. PPC was isolated from white cabbage using a saline solution containing hydrochloric acid (pH 1.5) at 37 °C for 4 h. PPC proteins were precipitated by aqueous 10% (m/v) trichloroacetic acid to yield the pectin-protein fractions PPC1 and PPC2. PPC was digested using 1,4-α-d-galacturonase, yielding the PPC6 fraction. Partial acid hydrolysis of PPC revealed the galacturonan fraction, PPC3, to be the core of the macromolecule. The purified PPC4 and PPC5 fractions were isolated from PPC by ion-exchange chromatography on DEAE-cellulose. ßG activity and its adsorption in the PPC fractions were studied in vitro. Crystalline cellulose was used as a control. This study found that the PPC3 fraction (the galacturonan core) does not adsorb ßG and does not affect its activity. The adsorption of ßG in the PPC samples is inversely proportional to the degree of methyl esterification of its carbohydrate component. The PPC4 and PPC5 fractions adsorb the highest proportion of ßG (51.2% and 54%, respectively). The stimulation of ßG enzyme activity is directly proportional to the protein content of the PPC sample. The PPC and PPC1 samples have the greatest ability to increase ßG activity (57.6% and 52.1%, respectively).

Structural studies of the pectic polysaccharide from Siberian fir (Abies sibirica Ledeb.).

The pectic polysaccharide named abienan AS-A was isolated from the wood greenery of Abies sibirica using dilute hydrochloric acid (pH 4.0) at 70°C. The structure of abienan AS-A was elucidated using sugar composition analysis, ion-exchange chromatography and partial acid hydrolysis followed by NMR spectroscopy. The linear region of abienan AS-A was shown to contain linear 1,4-α-D-galactopyranosyluronan partially substituted with methyl esters or 3-O-acetyl groups and rhamnogalacturonan blocks consisting of 1,4-α-D-galacturonan partially substituted with methyl ester groups and connected by 2-O-substituted α-rhamnopyranose residues. The branched region of abienan AS-A was found to be made of RG-I. The side chains of RG-I were shown to contain 1,4-ß-galactan and branched arabinan. Some 4-O-substituted ß-galactopyranose residues were shown to be attached to the 4-position of the 2-O-substituted α-rhamnopyranose residues of the RG-I backbone. The arabinan groups were made up of a 1,5-linked α-L-arabinofuranan backbone that was 3-O-, 2-O-, and 2,3-di-O-substituted with the terminal and 1,3-linked α-L-arabinofuranose residues.