Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device.

Numerous monosaccharides and oligosaccharides were derivatized at their reducing end groups with the fluorophore 2-aminoacridone. The resulting fluorescent compounds were separated by PAGE using two different buffer systems. One of these, a Tris borate buffer, enabled all of the fluorescent saccharide derivatives tested to be electrophoresed and various positional isomers, anomers, and epimers could be separated. The other system consisted of a discontinuous Tris-HCl/Tris-glycine buffer and enabled the electrophoresis of acidic, but not neutral, saccharide derivatives. The acidic and neutral saccharides could thus be distinguished unequivocally. The fluorescent labeling procedure was virtually quantitative and as little as 0.63 pmol could be detected photographically when gels were illuminated by uv light. When gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrates of glycoconjugates and other naturally occurring oligosaccharides.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Analyses of oligosaccharides by tagging the reducing end with a fluorescent compound. I. Application to glycoproteins.

The reducing end sugar of an oligosaccharide and 2-aminopyridine were linked by means of reductive amination with sodium cyanoborohydride. The fluorescent derivative of the oligosaccharide thus obtained, which had a positive charge, was subjected to two-dimensional paper electrophoresis. In the first direction, the sugar derivative moved according to its degree of polymerization, and in the second direction, it moved according to the structure of the borate complex. In this way fluorescent derivatives of saccharides were mapped on a sheet of paper. The method was applied to some known mono- and oligosaccharides and to the saccharides obtained by nitrous deamination of the oligosaccharide portions of glycoproteins (fetuin, Take-amylase A, and ovalbumin). The fingerprints thus obtained were characteristic of the chemical structures of the original oligosaccharides.     

The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis

The glycans of glycoconjugates mediate numerous important biological processes. Their separation and structural determination present considerable difficulties because of the small quantities that are available from biological sources and the inherent difficulty of analyzing the wide variety of complex structures that exist. A method for the analysis of reducing saccharides by PAGE that uses specific fluorophore labeling and is simple, rapid, sensitive, and readily available to biological researchers, has been developed. The method is known acronimically either as PAGEFS (PAGE of Fluorophore-labeled Saccharides) or in one commercial format as FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). In the PAGEFS method, saccharides having an aldehydic reducing end group are labeled quantitatively with a fluorophore and then separated with high resolution by PAGE. Two fluorophores, 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS) and 2-aminoacridone (AMAC), have been used to enable the separation of a variety of saccharide positional isomers, anomers, and epimers. Subpicomolar quantities of individual saccharides can be detected using a sensitive imaging system. Mixtures of oligosaccharides obtained by enzymatic cleavage from glycoproteins can be labeled and electrophoresed to yield an oligosaccharide profile of each protein. AMAC can be used to distinguish unequivocally between acidic and neutral oligosaccharides. Methods for obtaining saccharide sequence information from purified oligosaccharides have been developed using enzymatic degradation. Other applications and the potential of the system are described.     

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound ''high-mannose'' oligosaccharides.

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.     

Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products

A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.     

A heteroduplex method for detection of targeted sub-populations of bacterial communities

We describe a simple method, based on heteroduplex mobility analysis of 16S rDNA fragments, for targeted detection of sub-populations of bacteria within diverse microbial communities. A small (ca. 200 bp) polymorphic fragment of the bacterial 16S rRNA gene was amplified from sample DNA using universal primers. Sample products were hybridised with a fluorescently labelled fragment amplified from a selected 'reporter' organism representing the target group. The resulting products were resolved and the labelled heteroduplex pairs detected on non-denaturing gels using automated DNA detection technology. A model, based on analysis of samples with known 16S rDNA sequences, demonstrates that heteroduplex mobility is inversely correlated with genetic distance and that beyond 26% genetic difference, heteroduplex products are not detected. The utility of the method was tested by field studies in which stream biofilms could be characterised by heteroduplex profiles generated with heterotrophic and autotrophic reporter organisms representing target groups.     

Labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry

A well-known reaction of carbonyl compounds with phenylhydrazine has been applied to saccharides, providing increased sensitivity for mass spectrometric (MS) and ultraviolet (UV) detection during high-performance liquid chromatographic (HPLC) separations. After a simple derivatization procedure for 1 h at 70 degrees C and purification of the reaction mixture from excess reagent by extraction, the sugar derivatives were characterized by direct injection or on-line HPLC/electrospray ionization (ESI) and by matrix-assisted laser desorption/ionization (MALDI) MS. Because no salts are used or produced upon reaction, this procedure is very simple and suitable for the tagging of saccharides. The reaction allows for on-target derivatization and products are very stable. The derivatization procedure has been applied to commercially-obtained small saccharides and standard N-linked oligosaccharides. Lastly, hen ovalbumin N-glycans were detached enzymatically and characterized by MALDI-MS as their phenylhydrazone derivatives.     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.     

Identification of ketoses by use of their peracetylated oxime derivatives: A G.L.C.-M.S. approach

A method based on peracetylated oxime (PAKO) derivatives has been developed for rapid g.l.c.-m.s. survey of ketoses. This derivatization procedure (and the chromatographic analysis of these derivatives) is identical to one previously employed to identify aldoses by means of peracetylated aldononitrile (PAAN) derivatives. The production of chemically different derivatives from the aldoses and ketoses by the same derivatization procedure greatly simplifies the chromatographic separation of the derivatives of the ketoses from those of the aldoses, and also results in distinctively different, mass-spectral fragmentation-pathways for the two sets of derivatives. Both the electron-impact (e.i.) and ammonia chemical-ionization (c.i.) mass spectra of PAKO derivatives have been examined. Extensive differences between the fragmentation-pathways of the PAAN and the PAKO derivatives have been observed both by e.i.m.s. and ammonia c.i.m.s. The g.l.c.-m.s. of these PAKO derivatives, in conjunction with various, isotopic variants of the derivatization process, can yield extensive structural information with regard to the starting saccharides associated with the known, or unknown, g.l.c. peaks. The g.l.c. and mass-spectral properties of highly O-methylated PAKO derivatives of d-fructose are compared, and contrasted, to those of the PAKO derivatives of non-O-methylated saccharides. The chromatographic properties of derivatives of oligosaccharides that result from the PAAN-PAKO derivatization procedure have also been studied.     

Histological and biochemical parameters of Crocus sativus during in vitro root and shoot organogenesis

Content of malondialdehyde (MDA), proline, phenolics, and saccharides was analyzed during different developmental stages of in vitro root and shoot organogenesis in saffron. The highest content of MDA, proline, and phenolics was detected in nodular calli. Significant changes were also found in the content of polysaccharides, soluble saccharides, oligosaccharides, and reducing saccharides during developmental stages. Histological investigation of nodular calli showed meristematic zones with small and densely stained cells situated at peripheral zones of calli. The meristematic zones surrounded some vascular areas from which de novo organs originated. The parenchymatic cells of inner zones of calli converted to procambium cells that produced vascular tissues.     

The use of an amino acid analyzer for the rapid identification and quantitative determination of chitosan oligosaccharides.

The oligosaccharides of chitosan, glucosamine through chitohexaose, were separated and quantitated with the use of an amino acid analyzer. The oligosaccharides can be satisfactorilly resolved with a step gradient using two buffers, but resolution is improved with a linear buffer gradient. Using experimentally determined color factors, the method permits the rapid quantitation and identification of small amounts of chitosan oligosaccharides.     

Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.     

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.     

2-Aminopyridine—a label for bridging of oligosaccharides HPLC profiling and glycoarray printing

2-Aminopyridine derivatives of oligosaccharides (OS-AP) were printed onto microchips by two different ways. The first method is based on direct covalent insertion of OS-AP in polyacrylamide gel 3D chip. The second method is based on conversion of OS-AP into more reactive OS-aminoalditol followed by covalent printing onto NHS-activated glass slides. This approach extends the range of saccharides suitable for covalent printing due to availability of commercial OS-AP and easy high-performance liquid chromatography separation of glycoprotein N-chains in form of AP derivatives.     

Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan

Various oligosaccharides from hyaluronic acid, which have glucuronic acid or N- acetylglucosamine at the nonreducing terminal, were prepared by digestion with a combination of testicular hyaluronidase and beta- glucuronidase. These oligo saccharides were analyzed by negative-mode ion-spray mass spectrometry (MS) with an atmospheric pressure ion source. Introduction of collisionally activated dissociation tandem mass spectrometry (CAD-MS/MS) produced ions derived from cleavage of the glycosidic bonds, allowing the structure to be analyzed. The CAD- MS/MS spectrum showed an intense and characteristic fragment ion at m/z 193 for oligosaccharides having glucuronic acid at the nonreducing terminal. On the other hand, this ion was not observed in the spectra of oligosaccharides having N- acetylglucosamine at the nonreducing terminal. Therefore, the fragmentation pattern revealed by CAD-MS/MS provides useful information for distinguishing glucuronic acid and N- acetylglucosamine at the nonreducing terminal of oligosaccharides derived from hyaluronic acid and other glycosaminoglycans. This ion- spray CAD-MS/MS technique was also applied successfully to the characterization of glycosaminoglycans reconstructed by glycotechnology.      

Inhibition Pattern of Beet-Saponin on Amylose Formation by Potato Phosphorylase

By using the multiple ascent technique, the authors have resolved the first several oligosaccharides of the product of initial stage of potato phosphorylase action in both the absence and the presence of beet-saponin with maltotriose as primer. The resolved chromatogram was sprayed with a mixture of G-1-P and phosphorylase, followed by spraying with iodine solution to locate the spots in which starch synthesis occurred.

Multi-chain mechanism of amylose formation from maltotriose and the suppression of lengthening of amylose chain by beet-saponin in the lag stage of enzyme action could be shown on paper chromatogram. No saccharides other than amylose series were recognized in the case of phosphorylase inhibition by beet-saponin.     

Neutral, acidic, and basic derivatives of anthranilamide that confer different formal charge to reducing oligosaccharides

To facilitate the use of oligosaccharides as analytical tools in biological studies, we have designed, synthesized, and conjugated to maltosaccharides a novel series of homologous small fluorescent moieties that differ in formal charge. These moieties are amide derivatives of anthranilic acid: uncharged N-(2-aminobenzoyl)glycinamide (ABGlyAmide; 2), acidic N,N-dimethyl-N(')-(2-aminobenzoyl)ethylenediamine (ABGlyDIMED; 3), and basic N-(2-aminobenzoyl)glycine (ABGly; 1). Routes for synthesis and optimal reaction conditions for glycoconjugation by conventional reductive amination are presented, as is the compatibility of these adducts with common analytical and preparative chromatographic methods, including RP-HPLC and HPAEC-PAD. These novel anthranilic acid derivatives confer both fluorescence and defined charge to oligosaccharides, and so enhance the repertoire of chromatographic and analytical methods for which anthranilic acid can be used. Furthermore, because glucosaccharides have rigid solution structure, these small fluorescent adducts with different formal charge are ideal tools for molecular sizing studies of membrane pores.