Hans Grasdalen

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy☆

The composition and sequence of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogeneous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degree of acetylation of 50% were soluble at neutral pH.

Inhomogeneous polysaccharide ionic gels

Abstract Polysaccharide ionotropic gels formed by diffusion of calcium ions into solutions of sodium alginate or pectate exhibited various degrees of inhomogeneity, in the sense that the polymer concentration was much higher at the surface than in the center of the gels. This was in contrast to the uniform gels obtained when κ-carrageenan was dialyzed against potassium ions and gellan gum against potassium and lead ions. The non-uniform distribution of polymer in alginate and pectate depended on parameters such as polymer concentration and molecular size, and the concentration of the gel inducing ion in the outer reservoir. For alginate a high fraction of l -guluronate residues slightly increased the inhomogeneity, whereas the presence of non-gelling cations strongly decreased it. A theory, limited to diffusion of reactants in infinite long tubes, compared with relevant experiments, suggested that the gelation was controlled by the relative rate of diffusion of polymer and gelling cations and that the gelling in the absence of non-gelling cations was irreversible and stoichiometric. A new simple technique for generating homogeneous calcium alginate gels by diffusion was also demonstrated.

Gelation of gellan gum

Abstract The microbial polysaccharide, gellan gum, was studied in aqueous solution and in the gel state by osmometry, viscometry, light scattering, polarimetry and NMR and by measurements of gel strength and cation-exchange selectivity. In solutions of the tetramethylammonium (TMA) salt of the polymer, increasing concentrations of TMACl induced contraction, ordering, and association of the chains, as revealed by viscometry and changes in optical rotation. Chain ordering occurred at ionic strengths (I) between 0·005 and 0·05 ( m ), and light scattering indicated an elongated chain structure in 0·025 m TMACl. Gelation was dependent upon both ionic strength and the identity of the cation. For monovalent cations at I = 0·1 ( m ) , gel strength increased in the order: TMA + + + + + + For divalent cations the series was: Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ 2+ 2+ 2+ The lack of specificity among the alkaline-earth cations distinguished gellan gum markedly from other uronic-acid containing polysaccharides like alginate and pectin. Studies of cation-exchange equilibria likewise showed no selectivity within the alkali-metal or the alkaline-earth metal cations, but for all the divalent ions the affinity series: Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ 2+ 2+ 2+ was the same as the series for increasing gel strength. Measurements of optical rotation and NMR spectroscopy indicated that chain association and gelation were related phenomena. The results are interpreted as indicating that gelation occurs in two steps, namely, chain ordering and chain association.

13C-N.m.r. studies of the acetylation sequences in partially N-deacetylated chitins (chitosans)

Chitosans obtained under homogeneous conditions of N-deacetylation, with degrees of N-deacetylation between 46% and 94%, were depolymerised and their 125-MHz 13C-n.m.r. spectra have been interpreted. The sequence of 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN) residues influenced the chemical shifts, and the diad and triad frequencies have been calculated. Chitosans that were N-deacetylated under homogeneous and heterogeneous conditions gave values for the diad and triad frequencies that were consistent with a random arrangement of GlcN and GlcNAc residues.

Determination of the degree of esterification and the distribution of methylated and free carboxyl groups in pectins by 1H-N.M.R. spectroscopy

Abstract The 400-MHz 1 H-n.m.r. spectra of pectins with various degrees of esterification (d.e.), as obtained by methylation and subsequent partial saponification, have been measured. The d.e. was obtained from the intensity of the signal for H-5, which shifted downfield (∼3 p.p.m.) on esterification. Sequence-dependent shielding of H-5 and H-1 made it possible to determine the fractions of the four possible diads and the four galacturonate (G)-centred triads. The diad and triad frequencies obtained from the relevant peak-area ratios indicated that the action of alkali leads to pectic molecules in which free and methyl-esterified carboxylic acids are randomly distributed.

Positional distribution of ω3 Fatty acids in marine lipid triacylglycerols by high-resolution13C nuclear magnetic resonance spectroscopy

The positional distribution [α(1,3)-acyl and ß(2)-acyl] of ω3 fatty acids [18:4(n-3), 20:4(n-3), 20:5(n-3), 22:5(n-3) and 22:6(n-3)] in depot fat of Atlantic salmon (Salmo salar), harp seal oil and cod liver oil triacylglycerols has been examined by13C nuclear magnetic resonance (NMR) spectroscopy. The positional distribution data can be defined from the spectrum of the carbonyl (C1 carbon) and the methylene (C2 and glyceryl carbon) regions. In depot fat of Atlantic salmon and cod liver oil, docosahexaenoic acid (DHA) was concentrated in the ß-position of the triacylglycerides with 72.6 and 74.4%, respectively. Only 3.2% of DHA and 4.6% of eicosapentaenoic acid (EPA) were esterified to the ß-position of the triacylglycerides in harp seal oil. EPA is nearly randomly distributed in cod liver oil and muscle lipids of Atlantic salmon, with 37.8 and 39.7%, respectively, in the ß-position. In general, the13C NMR-derived data were in accordance with corresponding data reported in the literature obtained by conventional techniques.

NMR spectroscopy studies of the action pattern of tomato pectinesterase: generation of block structure in pectin by a multiple-attack mechanism☆

Abstract The enzymatic de-esterification of highly exterified poly( d -galacturonate) (degree of esterification, d.e. ≈ 0.92, dp n ≈ 15) was monitored by 1 H NMR spectroscopy. The enzymatic reaction resulted in a high content of homogeneous triads (GGG and EEE) demonstrating the production of a sequential structure. At high d.e. values, the enzymatic activity increased in proportion to the content of GE-diads. A multiple-attack mechanism was indicated by a relatively slow decrease of single Gs (EGE) in the sequential structure. Production of EGG-triads in great preference to GGE-triads pointed to a degree of multiple-attack greater than the effective number average block length of E-residues, ≈ 7–8, and, interestingly, the residue at the reducing end was de-esterified faster than that at the non-reducing end. These findings support the assumption that the enzyme attacks in alternating sequences (-GE-) and de-esterifies linearly preferentially towards the reducing end. Simulation-computed results are reported to demonstrate the difference between multiple-attack and multiple-chain mechanisms.

The use of neocarrabiose oligosaccharides with different length and sulphate substitution as model compounds for 1H-NMR spectroscopy

The high-field 1H-NMR spectra of various carrageenan oligosaccharides at room temperature are given. The assignments were faciliated by the use of proton double-quantum coherence (DQCOSY) and 1H-13C chemical shift correlation 2D NMR spectroscopy, and by comparing high-field 1H-NMR spectra of various 4-sulphated oligosaccharides of the neocarrabiose type. The effects of anomeric configuration on the 1H resonances on the same or neighbouring units are discussed. The 13C-NMR shift data are given for the tetrasaccharide of kappa-carrageenan.

Analysis of carrageenans by enzymic degradation, gel filtration and 1H NMR spectroscopy

Abstract Carrageenans from Furcellaria lumbricalis and Eucheuma gelatinae were degraded by κ-carrageenase from Pseudomonas carrageenovora . Higher and lower molecular weight fractions were separated by ethanol precipitation and further fractionated by GPC on serially connected Sephacryl S-100 and S-200, and BioGel P4, respectively, and investigated by 500 MHz 1 H NMR spectroscopy. Except for the lowest molecular weight oligosaccharide fractions from E. gelatinae , which contained di- and tetrasaccharides of κ-carrageenan, the lower molecular weight fractions of the carrageenans contained mixtures of neocarrabiose oligosaccharides with different content of d -galactose-4-sulphate. Both neocarrabiose and neocarrabiose-4-sulphate were detected on the non-reducing end. Varying with the source, the higher molecular weight fractions contained methyl, sulphate and other substitutions masking the general repeating backbone of neocarrabiose and neocarrabiose-4-sulphate.

Quantitative high-resolution 13C nuclear magnetic resonance of anserine and lactate in white muscle of Atlantic salmon (Salmo salar)

Quantitative 13C nuclear magnetic resonance (NMR) spectra of minced white muscle of Atlantic salmon (Salmo salar) were obtained without chemical pretreatment of the sample. The carbon in the metabolites anserine and lactate gave rise to sufficiently resolved signals in the 13C spectrum to permit estimation of the total concentration of these muscle constituents. The NMR data are in good agreement with corresponding data reported in the literature obtained by conventional methods, indicating that all of these metabolites are NMR visible. Information about the muscle pH was obtained from the pH-dependent 13C chemical shift of C14 and C16 in the histidine ring of anserine. Well-resolved 13C NMR spectra obtained from intact muscle of Atlantic salmon demonstrate that NMR will be a useful method to study postmortem changes occurring during storage of fish.