Smith Degradation Products Research Articles

Heterogeneous Components of Chitosans

The main objectives of the research were to compare the components of partially N-deacetylated chitins prepared identically from native chitin and a chitin regenerated from a heavily deacetylated chitosan. Additionally, to determine if any of the water-soluble components would serve as substrates in a study of a Chitinase isolated from soy bean hull. The brief heating of suspended chitins in 20% (w/w) NaOH resulted in similar degrees of N-deacetylation, the native chitin giving DAc 0.84 and the regenerated chitin DAc 0.79-0.72, with DAc indicating the proportion of glucosamine residues that are acetylated. Evidence for the nature of the hydrolysis of acetamido groups was provided by analyses of the water-soluble and -insoluble Smith degradation products. The water-soluble fraction derived from the native chitin comprised very small amounts of erythrityl N-acetyl glucosaminoside (GlcNAc1E), erythrityl N,N'-diacetyl chitobioside (GlcNAc2E), and erythrityl N,N',N''-triacetyl chitotrioside (GlcNAc3E), each identified by MALDI-TOF mass spectrometry of the butanoyl derivative. The water-insoluble products, as analyzed by light scattering detection method of their butanoyl esters and corrected for their composition, had a molecular weight (Mw) of 25 kDa, corresponding to about 120 N-acetyl glucosaminyl repeating residues (DPw), contrasting to that of 140 kDa with DPw of 680 for the parent chitin. Much of the decrease in the molecular weight of the polymer occurs by the loss of sugar residues by alkaline peeling at reducing terminals. For the regenerated chitin (DAc 1.0), prepared by N-reacetylation of a commercial chitosan (DAc 0.15), the resulting Smith products comprised erythritol and a series of N-acetyl glucosaminyl erythritol homologues of up to at least 39 N-acetyl glucosaminyl repeating residues, reflecting greater heterogeneity in the hydrolysis of acetamido groups along the polymer chain than what was seen for the native chitin. Of the water-soluble Smith products, GlcNAc5-7E were good substrates for chitinase isolated from soybean hull.

Analysis of fluorogenic Smith degradation products of 7-(1,3-disulfonaphtyl)amino-disaccharides for linkage position analysis of carbohydrates.

The linkage position of a glycosidic bond to the reducing-end residue of a pyridylamino (PA-) sugar can be determined sensitively by Smith degradation and HPLC [K. Omichi and S. Hase, (1994) J. Biochem. 115, 429-434]. With the aim of enhancing the sensitivity of this method of linkage position analysis to the fmol-level, use of the 7-(1,3-disulfonaphtyl)amino (DSNA-) group instead of the PA-group as a fluorescent tag was examined. Smith degradation of DSNA-disaccharides with a DSNA-hexose, DSNA-N-acetylglucosamine, or DSNA-N-acetylgalactosamine reducing-end residue was carried out. HPLC and FAB-MS of the fluorogenic Smith degradation products showed that the DSNA-group was stable under the Smith degradation reaction conditions, and that the reaction proceeded in a manner similar to that using PA-disaccharides to give the predicted products. Fluorogenic Smith degradation products specific to the glycosidic linkage position were well separated by reversed-phase HPLC, and were easily assignable by comparing the HPLC elution positions with those of standard compounds. The method was successfully applied to analyzing the structure of an N-linked sugar chain.

Antitumor activity and chemical modification of polysaccharides from niohshimeji mushroom, Tricholma giganteum.

Two chemical modification procedures, Smith degradation and formolysis processes, were used in an attempt to improve the antitumor activity of water-soluble and water-insoluble polysaccharides prepared from the fruiting body of niohshimeji, Tricholoma giganteum. The chemically modified products were examined for their antitumor effects on Sarcoma 180 solid tumor implanted in mice. The following results were as follows: Some Smith degradation products, i.e., O-R-Flo-c-beta and O-R-FA-2 prepared from water-soluble polysaccharides, and O-R-FII-1 and O-R-FIII-2-c from water-insoluble polysaccharides, had higher antitumor activities than the original polysaccharides. None of the formolysis products, F-Flo-a, F-W-Flo-a, F-FA-3, F-W-FA-3 from water-soluble polysaccharides, and F-FII-2, F-W-FII-2, F-FIII-2-b, and F-W-FIII-2-b prepared from water-insoluble polysaccharides had improved antitumor activities.

The structure of a novel polysaccharide produced by Bradyrhizobium species within soybean nodules

Certain strains of Bradyrhizobium japonicum and B. elkanii produce a polysaccharide within the root nodules of their legume host, soybean. These nodule polysaccharides (NPSs) were isolated and characeterized. The NPS produced by B. elkanii strains proved to be identical in glycosyl composition and linkages to the extracellular polysaccharide (EPS) of this species indicating that the NPS and EPS for B. elkanii have identical structures (W.F. Dudman, Carbohydr. Res., 66 (1978) 9–23), ▪ However, the structure of the NPS from B. japonicum proved to be quite different from that of its EPS. Methylation analysis of this NPS showed that it consists of 3-linked Gal, 3-linked Rha, 2,4-linked Rha, 4-linked Rha, and terminal 2- O-methyl GLcA in a 1:1:1:1:1 ratio. Stereochemical configurations of the glycosyl residues were determined by the preparation and analysis of trimethylsilyl (Me 3Si) (−)-2-butylglycosides. NMR spectroscopy (both 1H and 13C) showed that the Gal residue is α-linked, while all the other glycosyl residues are β-linked. Oligosaccharides produced by periodate oxidation-Smith degradation were purified, as were oligosaccharides produced by partial acid hydrolysis. Characterization of the Smith degradation products by methylation analysis, NMR spectroscopy, electrospray-mass spectrometry, and characterization of the partial acid hydrolysate oligsaccharides showed that the repeating oligosaccharide unit of the NPS has the structure, ▪.

A conformational study of the Smith degradation product of the Klebsiella K40 capsular polysaccharide by 1D NOESY and molecular mechanics calculations

A conformational study of the Smith degradation product of the Klebsiella K40 capsular polysaccharide by 1D NOESY and molecular mechanics calculations

Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

The structure of the O-specific polysaccharide of Citrobacter O16 containing glycerol phosphate.

The O-specific polysaccharide, obtained by mild acid degradation of Citrobacter O16 lipopolysaccharide, consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, glycerol and phosphate in the ratios 2:2:2:1:1. Selective cleavage of the polysaccharide was carried out by Smith degradation, N-deacetylation-deamination and dephosphorylation with 48% hydrofluoric acid, which was accompanied by unexpected splitting of one of the glycosidic linkages. The structures of the oligosaccharides thus obtained were established using 1H- and 13C-NMR spectroscopy, including one-dimensional NOE, two-dimensional rotating-frame NOE, homonuclear and heteronuclear 13C, 1H correlation spectroscopy, and, for the Smith degradation product, positive- and negative-ion-mode fast-atom-bombardment MS and MS/MS with collision-induced dissociation. On the basis of these data and the results of methylation analysis, it was concluded that the O-specific polysaccharide has the following repeating unit structure: [formula: see text]

The core structure of ginsenan PA, a phagocytosis-activating polysaccharide from the root of Panax ginseng.

Controlled Smith degradation and limited hydrolysis of ginsenan PA, the main phagocytosis-activating polysaccharide isolated from the root of Panax ginseng C. A. Meyer, were performed. The reticuloendothelial system-potentiating and anti-complementary activities of the degradation products were investigated. Methylation analysis of the primary and secondary Smith degradation products indicated that the core structural features of ginsenan PA include a backbone chain mainly composed of beta-1,3-linked D-galactose. Almost half of the galactose units in the backbone carry side-chains composed of beta-1,6-linked D-galactosyl residues at position 6. Further 3,6-branching of D-galactose units was observed in a part of the side-chains. alpha-L-Arabinose units are connected mainly to the core galactose moieties via position 6. Removal of most of the arabinose units had a considerable effect on immunological activity.

Arabinogalactan core structure and immunological activities of ukonan C, an acidic polysaccharide from the rhizome of Curcuma longa.

Controlled Smith degradation of ukonan C, a phagocytosis-activating polysaccharide isolated from the rhizome of Curcuma longa L., was performed. The reticuloendothelial system-potentiating, anti-complementary and alkaline phosphatase-inducing activities of ukonan C and its degradation products were investigated. Methylation analyses of the primary and secondary Smith degradation products and of a de-arabinosylated product indicated that structural features of the arabinogalactan core of ukonan C include a backbone chain composed of beta-1,3-linked D-galactose and beta-1,4-linked D-xylose. All of the galactose units in the backbone carry side chains composed of beta-1,6-linked D-galactosyl residues with or without terminal alpha-L-arabinose units at position 3. Ukonan C showed remarkable effects on both reticuloendothelial system-potentiating and alkaline phosphatase-inducing activities. Periodate oxidation caused a decrease in or disappearance of the immunological activities, but the controlled Smith degradation product having the arabinogalactan core structure of polysaccharide showed a pronounced effect on anti-complementary activity.

Constituents of the seed of Malva verticillata. VIII. Smith degradation of MVS-VI, the major acidic polysaccharide, and anti-complementary activity of products.

The controlled Smith degradation of MVS-VI, the major acidic polysaccharide having remarkable anti-complementary activity isolated from the seeds of Malva verticillata L., was performed. Methylation analysis of both the primary and the secondary Smith degradation products indicated that the core structural features of MVS-VI include a backbone chain composed of beta-1,3-linked D-galactose residues. The majority of galactose units in the backbone carry side chains composed of beta-1,3- and beta-1,6-linked D-galactosyl residues at position 6. The controlled Smith degradation products showed considerable anti-complementary activity.

Core structure of glycyrrhizan GA, the main polysaccharide from the stolon of Glycyrrhiza glabra var. glandulifera; anti-complementary and alkaline phosphatase-inducing activities of the polysaccharide and its degradation products.

The controlled Smith degradation and limited hydrolysis of glycyrrhizan GA, a representative polysaccharide with remarkable phagocytosis-enhancing activity isolated from the stolon of Glycyrrhiza glabra L. var. glandulifera Reg. et Herd. were carried out. Methylation analyses of the primary and the secondary Smith degradation products and of the limited hydrolysis product indicated that the core structural features of glycyrrhizan GA include a backbone chain composed of beta-1,3-linked D-galactose residues. Three-fifths of the galactose units in the backbone carry side chains composed of beta-1,3- and beta-1,6-linked D-galactosyl residues at position 6. Anti-complementary and alkaline phosphatase-inducing activities of the polysaccharide, periodate oxidation-reduction and the controlled Smith degradation products were investigated, and the controlled Smith degradation product showed significant activity.

The core structure of ukonan A, a phagocytosis-activating polysaccharide from the rhizome of Curcuma longa, and immunological activities of degradation products.

The controlled Smith degradation of ukonan A, a phagocytosis-activating polysaccharide isolated from the rhizome of Curcuma longa L., was performed. The reticuloendothelial system-potentiating, anti-complementary and alkaline phosphatase-inducing activities of ukonan A and its degradation products were investigated. Methylation analyses of both the primary and the secondary Smith degradation products indicated that the core structural features of ukonan A include a backbone chain mainly composed of beta-1,3-linked D-galactose, beta-1,4-linked D-xylose and alpha-1,2-linked L-rhamnose residues. All of the galactose units in the backbone carry side chains composed of alpha-L-arabino-beta-D-galactosyl or beta-D-galactosyl residues at position 6. Ukonan A has a remarkable effect on each of the three kinds of immunological activities. Periodate oxidation caused pronounced decrease or disappearance of the activities, but the controlled Smith degradation product having the core structure of polysaccharide showed considerable restoration of these activities.

The core structure and immunological activities of glycyrrhizan UA, the main polysaccharide from the root of Glycyrrhiza uralensis.

The controlled Smith degradation and limited hydrolysis of glycyrrhizan UA, the main phagocytosis-activating polysaccharide isolated from the root of Glycyrrhiza uralensis FISCHER, was performed. The reticuloendothelial system-potentiating, anti-complementary and alkaline phosphatase-inducing activities of glycyrrhizan UA and its degradation products were investigated. Methylation analyses of primary, secondary and tertiary Smith degradation products and of the limited hydrolysis product indicated that the core structural features of glycyrrhizan UA include a backbone chain composed of beta-1,3-linked D-galactose. All of the galactose units in the backbone carry side chains composed of mainly alpha-1,5-linked L-arabino-beta-1,6- or 1,3-linked D-galactose residues at position 6. Removal of the arabinosyl side chains caused a pronounced decrease in immunological activity.

Polysaccharides from Peptostreptococcus anaerobius and structure of the species-specific antigen

The cell-envelope antigens of Peptostreptococcus anaerobius were extracted from intact cells by autoclave or alkaline treatment. The purified species-specific antigen ( G) was identified among several polysaccharides obtained from the extracts by successive treatments with ribonuclease and pronase followed by ion-exchange and gel-filtration chromatography. G was investigated by 13C- and 31P-n.m.r. spectroscopy, titrimetry, elemental analysis, and gas-liquid chromatography. Oxidation of G with NaIO 4 followed by reduction with NaBH 4 and mild acid hydrolysis yielded the Smith degradation product of G ( GS). Treatment of G and GS with 48% HF gave the respective dephosphorylated products GF and GSF. The structures of GS, GF, and GSF were investigated by 13C-n.m.r. spectroscopy, methylation analysis, and gas-liquid chromatography-mass spectrometry. The principal constituents of G were 2-acetamido-2-deoxy- d-glucose ( d-GlcNAc), d-glyceric acid, and phosphate as a diester, in the ratio 2:1:1, and a minor amount of d-glucose (β- d-Glc p). GS contained d-GlcNAc, d-glyceric acid, glycerol, and phosphate in a 1:1:1:1 ratio. GF and GSF contained d-GlcNAc and d-glyceric acid in the ratios 2:1 and 1:1, respectively. A structure for the principal repeating unit of polymeric G compatible with the analytical data consists of α- d-Glc pNAc-(1→3)-α- d-Glc pNAc-(1→2)- d-glyceric acid units linked through C-6′-C-6″ phosphate diester bridges. This structure is novel for two reasons: ( a) unsubstituted glyceric acid residues occur as aglycons in the repeating structure, and ( b) phosphate diester bridges link nonanomeric glycose carbons in a non-nucleic acid polymer. The structural role of the minor amount of β- d-Glc p in G remains unknown.

The amino acid composition and quantitative sugar-amino acid relationships in sequential Smith-degradation products from gum talha (Acacia seyal Del.).

Data are presented for the amino acid and sugar compositions of gum talha (tahl), i.e. the exudate from Acacia seyal Del., and for four sequential Smith-degradation (SD) products (0.10, 0.19, 0.38, 1.60 and 2.22% N respectively). Gum talha, which is not permitted as a food additive, is liable to occur as a contaminant of shipments of legitimate gum arabic (Acacia senegal (L.) Willd.). The molar polysaccharide/protein ratio in the whole gum (113:1) decreased to 58:1, 27:1, 5.5:1 and 4:1 in the first, second, third and fourth SD products respectively. From previous studies, SD-IV is known to be a branched galactan; hydroxyproline, aspartic acid, serine, valine, proline, and leucine account jointly for 70% of its amino acids. The first SD eliminated large amounts of sugars (ca 50%, including all of the rhamnose and glucuronic acid present) but only very small amounts of amino acids (molar ratio 807/1). The second and third degradations eliminated all of the arabinose and 99% of the galactose originally present. The very small yield of the final SD product is an established characteristic of dextrorotatory Acacia exudates of the A. seyal type (Bentham's Gummiferae). Overall (four SD stages), 99.7% of the original sugars but only 91.3% of the amino acids were eliminated. Hydroxyproline accounts for ca 25% of the low amino acid content of A. seyal gum and each of its SD products; the proteinaceous enrichment of the branched galactan core also involves aspartic acid, glutamic acid and iso-leucine.(ABSTRACT TRUNCATED AT 250 WORDS)

The reactivity of galactose oxidase with snail galactans, galactosides and d-galactose-composed oligosaccharides

The enzymic oxidation of snail galactans, of their first and second Smith degradation products and of some structurally related polysaccharides was studied. Lymnaea stagnalis galactan after one cycle of Smith degradation reacted best and native Helix pomatia galactan was almost inactive. Investigations on the structural requirements for oligosaccharides to bind to galactose oxidase showed that the branched tetrasaccharide, Gal-β-1 → 6-[Gal-β-1 → 3]-Gal-β-1 → 1 l-Gro, in the terminal nonreducing position was the most complementary structure in the native galactan to associate with the enzyme. All nitrophenyl α-galactosides reacted better, and the ortho-form was 10-times more potent compared with this tetrasaccharide, indicative of the involvement of a hydrophobic region in binding. However, the β-linked isomers were only equally or less reactive than galactose. The enzymic oxidation determined colorimetrically by transferring the peroxide formed to o-dianisidine ceased at a maximum typical for each substrte and independent of the reaction time. When the absolute turn-over rates of ortho- and para-nitrophenyl α-galactoside and for the β-isomer were determined by HPLC, it could be demonstrated that the oxidation had not finished at the maximum of the colour reaction, but proceeded until the substrate was consumed. The initial speed of the colour reaction paralleled the absolute oxidation rate.

Fine structure of the arabinogalactan-protein from Lolium multiflorum

The extracellular arabinogalactan-protein from suspension-cultured cells of Lolium multiflorum (ryegrass) was purified in a single step by affinity chromatography of the culture medium on myeloma protein J539-Sepharose. The product obtained after two Smith-degradations had an apparent molecular size corresponding to that of galactoheptaose and was mainly (1→3)-linked. The Smith-degradation products were essentially monodisperse with respect to molecular size, indicating that periodate-oxidisable residues were distributed regularly along the polysaccharide chains. The polysaccharide chains in the arabinogalactan-protein were probably attached to hydroxyprolyl residues of the protein core via O-glycosylic linkages. No carbohydrate appears to be linked to serine or threonine residues. No oligo-arabinosides linked to hydroxyproline could be detected.

Arabinogalactan-protein components of Acacia tortilis GUM

The gum exudate from Acacia tortilis. its Smith-degradation products, and the partially hydrolysed gum are associated with protein. The presence of periodate-immune arabinosyl chains which protect otherwise vulnerable galactosyl residues is the cause of only limited breakdown of the gum during Smith degradation. Removal of the arabinosyl groups followed by Smith degradation results in depolymerisation to give short galactose-containing chains, mainly of uniform size. A molecular representation of the core structure, based upon these results and those of methylation analysis, is given.

Structural investigations of the galactan of the snail Lymnaea stagnalis

A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d-galactose and 0.9% of nitrogen, but neither l-galactose nor phosphate groups. The [α] D 20 values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra- O-methyl- and 2,4-di- O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri- O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β- d-Gal-(1→1)- l-Gro, β- d-Gal-(1→3)-β- d-Gal-(1→1)- l-Gro, β- d-Gal-(1→6)-β- d-Gal-(1→1)- l-Gro, β- d-Gal-(1→6)- d-Gal-β- d-Gal-(1→3)-β- d-Gal-(1→1)- l-Gro, β- d-Gal-(1→3)-[β- d-Gal-(1→6)]-β- d-Gal-(1→1)- l-Gro, β- d-Gal-(1→3)-β- d-Gal-(1→6)-β- d-Gal-(1→1)- l-Gro, and β- d-Gal-(1→3)-β- d-Gal-(1→3)-β- d-Gal-(1→1)- l-Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.

Analysis of the common polysaccharide antigens from the cell envelope of Clostridium perfringens type A

The major, common antigen of Clostridium perfringens type A, isolated and purified independently from three selected strains (Hobbs 5, Hobbs 9, and Hobbs 10), is composed of equimolar amounts of 2-acetamido-2-deoxy- d-mannose (ManNAc) and 2-acetamido-2-deoxy- d-glucose (GlcNAc). The purified antigen gave a strong immunoprecipitin line by double immunodiffusion in gel. Smith degradation of the major, common antigen caused decomposition of all of the GlcNAc, without concomitant loss in ManNAc, or a perceptible change in serological activity. Therefore, the serological activity of the major, common antigen depended solely on the presence of ManNAc. Data obtained by the 13C-n.m.r.-spectral analysis of the Smith-degradation product revealed that it was a linear-backbone polysaccharide analogous to a Rhodotorula glutinis mannan, but composed of pairs of 2-acetamido-2-deoxymannopyranosyl residues alternately linked β-(1→3) and β-(1→4). The one-bond, carbon-hydrogen coupling-constant of 162 Hz for both anomeric centers was consistent with the proposed β-linkages. A similar, 13C-n.m.r.-spectral analysis of the native, common antigen indicated that the GlcNAc residues were randomly connected to three of the four hydroxyl groups not already involved in linking the ManNAc backbone, the 4-hydroxyl group being the exception. A second, serologically inactive, polysaccharide composed of rhamnose, GalNAc, and galactose was identified, but not obtained in homogeneous state. The rhamnosyl residues were probably situated as nonreducing antennae, as they were quantitatively removed by Smith degradation without concomitant decomposition of the polymeric structure of the remaining residues.