John W. Redmond

A GENERAL APPROACH TO DESALTING OLIGOSACCHARIDES RELEASED FROM GLYCOPROTEINS

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and β-eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.

Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii

Rhizobium trifolii mutants containing Escherichia coli lac gene fusions to specific nodulation (nod) genes were used to characterise phenolic compounds secreted from the roots of white clover (Trifolium repens) plants. These compounds either had stimulatory or inhibitory effects upon the induction of the nod genes. The stimulatory compounds were hydroxylated flavones and the most active compound was 7,4′‐dihydroxyflavone. The inhibitory compounds present in white clover root exudates were umbelliferone (a coumarin) and formononetin (an isoflavone). Transcriptional activation of nod gene promoters in response to short exposures (3 h) of 7,4′‐dihydroxyflavone was growth phase dependent; cells in early log phase were highly responsive to flavone additions in vitro and nod gene induction could be detected within 20 min of exposure at 5 x 10−7 M. Cells in other growth phases were generally unresponsive. A 10‐fold molar excess of umbelliferone to 7,4′‐dihydroxyflavone resulted in complete inhibition of nod gene induction. Some commercially‐obtained flavones were found to have weak stimulatory activity but could also inhibit nod gene induction by more effective stimulatory compounds. Strong stimulatory and inhibitory compounds all possessed a 7‐hydroxy moiety and showed other structural similarities. This suggested that there was one binding site for these compounds. Because the response to these compounds was rapid, we propose that these phenolics act at the bacterial membrane or that an active uptake system is involved.

Alteration of the Effective Nodulation Properties of a Fast-growing Broad Host Range Rhizobium due to Changes in Exopolysaccharide Synthesis

Summary Strain NGR234 is a broad host range Rhizobium which can effectively nodulate a broad spectrum of legumes. Ninety mutants with altered exopolysaccharide production were isolated after strain NGR234 was subjected to transposon Tn5 mutagenesis. These mutants were classified on the basis of their physiological properties into 9 groups. Their symbiotic porperties were tested on four legumes which form spherical (determinate) nodules ( Macroptilium atropurpureum, Desmodium intortum, Desmodium uncinatum and Lablab purpureus ) and on the legume Leucaena leucocephala which forms cylindrical (indeterminate) nodules. On these plants strain NGR234 forms nitrogen-fixing nodules (Nod + Fix + ). The results from the testing of the various mucoid-defective mutants on these plants show that it is possible to alter the synthesis of the surface polysaccharides of strain NGR234 and produce a narrow host range Nod + Fix + Rhizobium .

Amino acid analysis using derivatisation with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography

A simple procedure for the precolumn derivatisation of amino acids with 9-fluorenylmethyl chloroformate and a liquid chromatographic method for the separation of the derivatives with fluorimetric detection in the picomole range are reported. The procedure does not involve a solvent extraction and gives single, stable derivatives of the common protein amino acids. The method has been demonstrated on hydrolysates of proteins and peptides.

Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate

A rapid and sensitive fully automated method for the determination of primary and secondary amino acids in different matrices is described. Amino acids are derivatized with 9-fluorenylmethyl chloroformate using an automated precolumn derivatization technique. Data are presented to show that the technique is both reproducible and highly sensitive. Applications of the technique are presented, including the analysis of peptide and protein hydrolysates and the profiling of free amino acids in physiological fluids.

The structure of the O-antigenic side chain of the lipopolysaccharide of Vibrio cholerae 569B (Inaba)

Mineral acid hydrolysis of the lipopolysaccharide from Vibrio cholerae 569B (Inaba) gives an oligosaccharide fraction which was shown, by use of 13C NMR and chemical methods, to be a regular alpha-(1 leads to 2) linked chain of D-perosamine (4-amino-4,6-dideoxy-D-mannose) units. This chain represents the O-antigen of the lipopolysaccharide, in which the amino functions are acylated with 3-hydroxypropionyl groups. The chromatographic properties of some hydroxamic acids are described and used to characterize these acyl groups.

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. I. Phosphorus magnetic resonance spectra

Abstract The 31P nuclear magnetic resonance spectrum of lipopolysaccharide from Salmonella minnesota R595 shows that the p K a and chemical shift values of the phosphate groups are very sensitive to changes in molecular structure. The presence of 3′ and / or 6′ substituents can be determined from the spectra of the 4′-phosphates and the type of 4′ or 6′ substituent affects the phosphates. When cells are grown in nutrient broth, the 4′ position in the lipopolysaccharide is substituted with as much as 91% aminoarabinose phosphate, with the remaining 9% as monophosphate (with 3′ acyl substituent). When L.B. medium is used, 52% of the 4′ position is substituted with phosphomonoester (with accompanying 3′-acyl group), with smaller amounts of monoester (with no 3′ substituent) and phosphodiester. Phosphate substitution at the 1 position is incomplete when cells are grown in nutrient broth. A detailed analysis is made of the chemical and physical parameters which influence p K a and chemical shift of phosphates. The conversion of 4′-phosphodiester to 4′-phosphomonoester and 4′-dephospho products with different hydrazine treatments was also studied.

The structure of the exopolysaccharide from Rhizobium sp. strain ANU280 (NGR234)

Abstract The structure of an exopolysaccharide from Rhizobium sp. strain ANU280 (derivative of the broad host-range strain NGR234) has been determined. Fragments generated by partial acid hydrolysis were fractionated by sequential ion-exchange ang gel chromatography, and their structures were assigned by 13 C-n.m.r. spectroscopy. Extensive overlap of structure between the fragments, together with the results of periodate oxidation and colorimetric analyses, permitted assignment of the nonsaccharide repeating-unit shown below. The terminal galactosyl group carries a 4,6- O -(1-carboxyethylidene) group and, probably, an acetyl group.

The reactions of 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) in dilute acid

Abstract On heating in dilute acid, 3-deoxy- d -manno-oct-2-ulosonic acid (KDO) is converted into 2,7-anhydro-3-deoxy-α- d -manno-2-octulofuranosonic acid and 5-( d -erythro-1,2,3-trihydropropyl)-2-furoic acid. The former is unreactive to periodic acid-thiobarbituric acid and to semicarbazide, and its formation explains the depressed estimates of KDO in lipopolysaccharides. Formation of the furoic acid can lead to high estimates using the semicarbazide assay. Neither product can be formed from 5-O-glycosyl-KDO.

Synthesis of methyl pyranosides and furanosides of 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) by acid-catalysed solvolysis of the acetylated derivatives

Treatment of methyl 2,4,5,7,8-penta-O-acetyl-3-deoxy-alpha-D-manno-oct- 2-ulopyranosonic acid, or its methyl ester, with refluxing methanolic 0.1 M hydrogen chloride for 16 h gave 95% of methyl (methyl 3-deoxy-alpha-D-manno-oct-2-ulopyranosid)onate. Acetylation of the methyl ester of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) gave mainly methyl 2,4,6,7,8-penta-O-acetyl-3-deoxy-alpha,beta-D-manno-oct-2-ulofuranoso nate. Treatment of this mixture with methanolic 0.02 M hydrogen chloride at room temperature gave methyl (methyl 3-deoxy-alpha, beta-D-manno-oct-2-ulofuranosid)onate and the corresponding 4-acetates which were isolated by reverse-phase column chromatography of their 7,8-O-isopropylidene derivatives. Confirmation of the position of the isopropylidene group was obtained by acetylation to give methyl (methyl 4,6-di-O-acetyl-3-deoxy-7,8-O-isopropylidene-alpha,beta-D-manno-oct-2-ul ofuranosid)onate. The furanose anomers were differentiated primarily by J3,4 values (alpha approximately 6.1 Hz, beta approximately 2.2 Hz). The anomeric configuration in the furanose series has been assigned on the basis of optical rotation.