Allene Jeanes

A new modification of the carbazole analysis: Application to heteropolysaccharides

Abstract The carbazole analysis when run at 55° in the presence of borate gives increased sensitivity for d -galacturonic, d -mannuronic, and l -guluronic acids, while greatly decreasing that of d -glueuronic acid. Comparison of values from these reaction conditions with values from other modifications of the carbazole analysis reveals a high degree of specificity of the reaction for each acid under any defined set of conditions. This specificity is of value for identification of unknown uronic acids. The new modification is especially useful for determination of d -mannuronic acid in heteropolysaccharides.

Six unusual dextrans: methylation structural analysis by combined g.l.c.—m.s. of per-O-acetyl-aldononitriles

Abstract Six bacterial dextrans from NRRL strains Leuconostoc mesenteroides B-1299, B-1303. B-1355, and B-1399; Streptobacterium dextranicum B-1254; and A g.l.c. procedure permitted, for the first time. separation of the 2,3,4- from the 2.3.6-tri- O -methyl derivative of d -glucose. Deuteriomethyla

Heterogeneity in dextran preparations

Abstract 1. 1. Three types of heterogeneity have been demonstrated in the polysaccharide products from many dextran-producing bacteria. These are gross heterogeneity in the culture, and both size and structural heterogeneity in the major fermentation product, dextran. 2. 2. Molecular size, physically and/or chemically bound impurities, or chemically bound fructose were responsible for the gross differences between the minor components of the dextran culture and the major component, dextran. 3. 3. Structural heterogeneity appears to be a normal occurrence in dextrans. The frequency and extent to which this heterogeneity occurs increases with the percentage of non-1,6-linked units. 4. 4. Analytical and preparative fractional precipitation methods have been established for studying the heterogeneity in dextrans. Application of these methods to five typical dextrans permits a tentative interpretation of the extent and nature of the heterogeneity in dextran preparations from many different bacterial strains. 5. 5. Information on the heterogeneity of the dextran component itself is essential for the characterization and utilization of dextrans. 6. 6. Examination of dextran products derived from progeny of individual cells of two strains which produce heterogeneous dextrans has failed to show the presence of mutant or variant cells.

Characterization and properties of the phosphomannan from Hansenula hostii NRRL Y-2448

Abstract The phosphorylated mannan produced in good yield from glucose by the bisexual diploid yeast, Hansenula holstii NRRL Y-2448, appears to be the first significantly phosphorylated polysaccharide to be obtained from a yeast or a nonpathogen. Isolated as the potassium salt, this water-soluble polysaccharide derivative has constituents in the proportion, d -mannose:phosphorus:potassium::5:1:1. The product has [ α ] D 25 + 103 ° (in 0.1 M potassium chloride), M N (reducing power) 103,000 ± 10,000, M W (light scattering) of the order of 16 × 10 6 , S 20, w = 44, and an unusually homogeneous molecular distribution for an unfractionated, native polymer. The single titration equivalence-point of the polyacid at pH 7.2 indicates a phosphodiester structure. Weak acid liberates secondary hydrogen ions and causes molecular degradation; dilute alkali appears to cause no structural change. Aqueous solutions show exceptional resistance to microbial attack. The brilliantly clear aqueous solutions have properties characteristic of a plastic, thixotropic, polyelectrolyte. The viscosity-concentration curve shows a viscosity maximum of 2500 cp. at 1.5% polysaccharide concentration and a minimum of 1700 cp. at 3%. At suitable concentrations of phosphomannan and borax, complexing and cross-linking occur; the presence of potassium chloride augments these effects.

Structural analysis of leuconostoc dextrans containing 3-O-α-d-glucosylated α-d-glucosyl residues in both linear-chain and branch-point positions, or only in branch-point positions, by methylation and by 13C-N.M.R. spectroscopy

Abstract It had been established by methylation-structural analysis that dextran fraction S from Leuconostoc mesenteroides NRRL B-1355 has two types of α- d -glucopyranosyl residues that are linked through O-3, i.e. , 35% of the residues carry a (1→3)-bond, and ∼10% carry a (1→6)-bond in addition to a (1→3)-bond. Two similarly constituted dextrans have now been identified by methylation-structural analysis, namely, the S-type fractions from L. mesenteroides strains NRRL B-1498 and B-1501. The S-type fractions from L. mesenteroides strains B-1355, B-1498, and B-1501 are structurally differentiated from the α- d -glucans (characteristically insoluble) of certain cariogenic Streptococci which also contain both 3- O - and 3,6-di- O -substituted α- d -glucopyranosyl residues. 13 C-N.m.r. spectra have been recorded at 90° for both the S- and L-type fractions of strains B-1355, b-1498, and B-1501. The L-type fractions have a low degree of branching through 3,6-di- O -substituted α d -glucopyranosyl residues, but no 3-mono- O -substituted residues. (Dextran fraction S of Streptococcus 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection of glass columns (2 mm i.d. x 1.23 m) was employed for all such chromatography. The 13 C-n.m.r. conditions and methods for preparation of dextran samples have been described(su4). In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the lock signal.

Determination of the composition of uronic acid mixtures.

Abstract A method has been developed to determine the relative proportions of three uronic acids in a compound or mixture, which uses data obtained from analysis of samples by the carbazole method under four different reaction conditions. The method works equally well with synthetic mixtures and natural products, such as alginates.

High-temperature enhancement of 13C-N.M.R. chemical-shifts of unusual dextrans, and correlation with methylation structural analysis

Abstract Dextran fractions from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and Streptobacterium dextranicum B-1254 were examined by 13 C-n.m.r. spectroscopy at 34 and 90°, and by methylation structural analysis. The native, structurally homogeneous dextran from L. mesenteroides NRRL B-1402 was also examined. The data allow correlations to be made between the structure and physical properties of the S (soluble) and L (less-soluble) fraction pairs of dextrans B-742, B-1254, B-1299, and B-1355. For the dextrans under consideration here, increasing solubility of the dextran (both in water and in aqueous ethanol) was found to correlate with decreasing percentages of α- d -(1→6)-linked d -glucopyranosyl residues. Both the diagnostic nature of the 70–75-p.p.m. spectral region with regard to type of dextran branching, and the increase in resolution of the polysaccharide spectra at higher temperatures, have been further confirmed.

Reaction of sodium hypochlorite with amines and amides: A new method for quantitating amino sugars in monomeric form☆

Abstract Free and N-acetylated hexosamines can be determined spectrophotometrically by a three-stage assay: chlorinating the amide or amine with NaOCl; reducing unreacted NaOCl with NaNO2; and reacting chloroamide (-amine) with amylose-KI reagent to produce the blue amylosetriiodide complex, measured at 615 nm. Distinctive behavior of the common 2-amino-2-deoxy- d -hexoses and their 2-acetyl derivatives allows three types of measurements to be made: (a) identification and differentiation by characteristic behavior (assay individually in primary cacodylate buffers over the pH range 6.0–9.5, then adjust pH to 5.5 with secondary phthalate buffer and remeasure the color); (b) assay individually in a single buffer at the optimum pH; and (c) assay differently admixed 2-amino-2-deoxy- d -hexose · HCl and 2-acctamindo-2-deoxy- d -hexose (assay in primary buffer at pH 9.0–9.5 gives measure of only HexNAc (no reaction with ManNAc), then adjust pH to 5.5 with secondary buffer to measure additional color that results only from HexN · HCl).

Structural analysis of insoluble d-glucans by fourier-transform, infrared difference-spectrometry: correlation between structures of dextrans from strains of leuconostoc mesenteroides and of d-glucans from strains of streptococcus mutans

Abstract Fourier-transform infrared (F.t.-i.r.) difference-spectra have been recorded, relative to a water-soluble dextran of low degree of branching, for (a) dextrans from Leuconostoc mesenteroides NRRL B-742 (the L fraction) and NRRL B-1149 (the A fraction), (b) d -glucans from Streptococcus mutans KR-1 and OMA 176, and (c) the controls of amylose, cellulose, nigeran, and pseudonigeran. Confirmation has been obtained for the presence, in the spectra of the relatively insoluble dextrans and d -glucans, of a previously recognized, characteristic absorbance at 822 cm−1, and the correlation of this band with contiguous, linearly (1 → 3)-linked, α- d -glucopyranosyl residues, to which polymer insolubility (and cariogenic properties) has been ascribed. This analytical method allows the mole percent of the contiguously linked 3-mono-O-substituted α- d -glucopyranosyl residues to be quickly and non-destructively established in solid-state samples, when employing weights of polysaccharides in the microgram range. The wavenumbers and intensities of other bands observed in the F.t.-i.r. difference-spectra of d -glucans containing (1→4)- d -linkages are also discussed.

Reaction of sodium hypochlorite with amines and amides: A new method for quantitating polysaccharides containing hexosamines

Abstract The NaOCl-amylose-KI spectrophotometric method for determining free and N -acetylated amino sugars has been applied to the analysis of PS Y-6272, which is composed of N -acetylglucosamine and N -acetylglucosaminuronic acid and produced extracellularly by the black yeast strain NRRL Y-6272 (6). PS Y-6272, as well as other amino sugar-containing polysaccharides such as chondroitin sulfate A, colominic acid, heparin, and hyaluronic acid, can be assayed readily in the range of 0.04 to 0.30 μmole N without previous depolymerization. Studies of the effects of pH and reaction time and temperature, carried out on these glycosaminoglycans as well as on various glucosamine derivatives and oligosaccharides, indicate that the color yield is influenced by the identity of the amino sugars present as well as by the position and anomeric type of linkages. However, since the color yield accords well with the nitrogen content of a particular amino sugar-containing polysaccharide, unknown quantities in solutions or column effluents can be determined readily and accurately by reference to calibration curves of the individual polysaccharide.