A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion.

The 13C-n.m.r. titration shifts of the alpha-methylene group of N-alkylated imidazoles are shown to be a sensitive probe of the ionization of the imidazolium ion. The 13C-n.m.r. titration shifts of both the intact and denatured/autolysed 2-13C- and 1-13C-enriched trypsin-7-amino-3-benzyloxycarbonylamino-1-chloroheptan-2-one (Z-Lys-CH2Cl) complexes are compared. The titration shift for the denatured/autolysed complex confirms that this ionization is due to deprotonation of the N-alkylated imidazolium ring of histidine-57. In the intact trypsin-inhibitor complex the titration shift due to the 1-13C-enriched carbon is anomalous. We conclude that this titration shift cannot arise solely from the ionization of the imidazolium ion of histidine-57 and that the pKa of the imidazolium ion of histidine-57 is raised in the trypsin-inhibitor complex. The relevance of these studies to the mechanism of action of the serine proteinases is discussed.     

Very-high-field n.m.r. studies of bovine lung heparan sulphate oligosaccharides produced by nitrous acid deaminative cleavage. 13C-n.m.r. study of methylene resonances: degree and positions of C-6 sulphation.

Oligosaccharides with the general structure UA-(GlcNAc-GlcUA-)m-aManOH (m = 1-5) (where UA represents uronic acid, GlcNAc N-acetylglucosamine, GlcUA glucuronic acid and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by nitrous acid deaminative cleavage followed by reduction. Analysis of the methylene signals in the 100 MHz 13C-n.m.r. spectrum of the tetrasaccharide (m = 1) shows that, whereas the extent of C-6 O-sulphation in the GlcNAc is approx. 65%, in the aManOH [formerly a GlcNSO3 (N-sulphoglucosamine) residue in the parent heparan sulphate] it is only approx. 10%. In the higher oligosaccharides (m = 2-5) the gross extent of C-6 O-sulphation of GlcNAc residues falls systematically with increasing oligosaccharide size, whereas that in the aManOH residues remains below 10%. There is also evidence that the C-6 O-sulphation of the GlcNAc residues is confined to the GlcNAc residue adjacent to the non-reducing terminal uronic acid residue. It is therefore tentatively proposed that the GlcNAc in the sequence -GlcNSO3-UA-GlcNAc- might be a favoured substrate for the 6-O-sulphotransferase. It is concluded that in the low-sulphated heparan sulphates GlcNSO3 residues that do not occur in (GlcNSO3-UA-)n blocks tend to have a significantly smaller extent of C-6 O-sulphation than do GlcNAc residues that occur in -GlcNSO3-UA-GlcNAc-GlcUA-GlcNSO3-sequences.     

Chondroitinase ABC digestion of dermatan sulphate. N.m.r. spectroscopic characterization of the oligo- and poly-saccharides.

Dermatan sulphates, in which iduronate was the predominant uronate constituent, were partially digested by chondroitinase ABC to produce oligosaccharides of the following structure: delta UA-[GalNAc(4SO3)-IdoA]mGalNAc(4SO3) [where m = 0-5, delta UA represents beta-D-gluco-4-enepyranosyluronate, IdoA represents alpha-L-iduronate and GalNAc(4SO3) represents 2-acetamido-2-deoxy-beta-D-galactose 4-O-sulphate], which were fractionated by gel-permeation chromatography and examined by 100 MHz 13C-n.m.r. and 400/500 MHz 1H-n.m.r. spectroscopy. Experimental conditions were established for the removal of non-reducing terminal unsaturated uronate residues by treatment with HgCL2, and reducing terminal N-acetylgalactosamine residues of the oligosaccharides were reduced with alkaline borohydride. These modifications were shown by 13C-n.m.r. spectroscopy to have proceeded to completion. Assignments of both 13C-n.m.r. and 1H-n.m.r. resonances are reported for the GalNAc(4SO3)-IdoA repeat sequence in the oligosaccharides as well as for the terminal residues resulting from enzyme digestion and subsequent modifications. A full analysis of a trisaccharide derived from dermatan sulphate led to the amendment of published 13C-n.m.r. chemical-shift assignments for the polymer.     

Structural features of a water-soluble arabinoxylan from the endosperm of wheat.

A cold-water-soluble wheat-endosperm arabinoxylan consisting of a backbone of (1----4)-linked beta-D-xylopyranosyl residues that are variously unsubstituted, and 3- or 2,3-substituted with single alpha-L-arabinofuranosyl groups, was subjected to 1H-n.m.r. spectroscopy. The results of 2D homonuclear Hartmann-Hahn and 1D 1H-n.m.r. spectroscopy allowed the identification of 3- and 2,3-substituted xylose residues, each with adjacent unsubstituted xylose residues, and also substituted xylose residues with a substituted xylose residue as a neighbour. The 1H-n.m.r. data were correlated with 13C-n.m.r. data by means of a 13C-1H 2D proton-detected heteronuclear multiple-quantum correlation experiment, which showed that only different types of branching (i.e., 3- and 2,3-) can be identified by the 13C-n.m.r. data.     

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus, contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and ¹³C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β-d-galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d-galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.     

Immunochemical examination of the Pseudomonas aeruginosa glycocalyx: a monoclonal antibody which recognizes L-guluronic acid residues of alginic acid

Mice immunized with Formalin-fixed mucoid Pseudomonas aeruginosa cells developed an immune response directed, in part, towards the P. aeruginosa glycocalyx. The polyclonal mouse sera produced good immunofluorescent staining of the P. aeruginosa glycocalyx and cell surface. A library of 250 hybridoma cell lines which produced monoclonal antibodies directed against P. aeruginosa was established. Twelve clones (4.8%) produced antibody which reacted with alginate in an enzyme-linked immunosorbent assay (ELISA). Clone Ps 53 was chosen for further study, cloned, and an ascites tumor established. Clone Ps 53 was chosen for further study because the antibody produced demonstrated a specificity similar to that of a recently isolated heparin--rat-lung lectin which recognizes alginates of the Homma nontypable P. aeruginosa strains. The Ps 53 clone produced an immunoglobulin M which reacted with P. aeruginosa alginate and produced good immunofluorescent staining of the P. aeruginosa glycocalyx. The Ps 53 monoclonal antibody has an apparent specificity for L-guluronic residues in ELISA. Competitive binding studies with various alginates and monosaccharides suggest that the C6 carboxyl group of uronic acids are recognized by the antibody and that the antigen-binding site is fairly large and may recognize a particular sequence or epitope of alginic acid which is rich in L-guluronic acid. The Ps 53 monoclonal antibody did not react uniformily with all P. aeruginosa alginates but did react with all of the alginates of the Homma nontypable strains tested, suggesting that acetylation or various modifications found in P. aeruginosa alginates may interfere with antibody binding and define specific epitopes. The Ps 53 antibody also reacted with purified outer membrane, indicating that some alginate or L-guluronic acid is intimately associated with outer membrane.     

DL-apiose substituted with stable isotopes: synthesis, n.m.r.-spectral analysis, and furanose anomerization

The branched-chain pentose DL-apiose has been synthesized in good yield by a new and simple chemical method that can be adapted to prepare (1-13C)-, (2-13C)-, (1-2H)- and/or (2-2H)-enriched derivatives. N.m.r. spectra (1H- and 13C-) have been interpreted with the aid of selective (13C)- and (2H)-enrichment, and 2D and 13C[13C]-n.m.r. spectra. The solution composition of DL-(1-13C)apiose in 2H2O, determined by 13C-n.m.r. spectroscopy, has been found to differ from that determined previously by 1H-n.m.r. spectroscopy. Several 13C-1H and 13C-13C couplings have been measured and interpreted in terms of apiofuranose ring conformation. Ring-opening rate-constants of the four apiofuranoses [3-C-(hydroxymethyl)-alpha- and -beta-D-erythrofuranose, and 3-C-(hydroxymethyl)-alpha- and -beta-L-threofuranose] have been determined by 13C-saturation-transfer n.m.r. spectroscopy, and compared to those obtained previously for the structurally related tetrofuranoses.     

Purification and characterization of cytosolic aldolase from carrot storage root.

The time courses of incorporation of 13C from 13C-labelled glucose or acetate into cerebral amino acids (glutamate, glutamine and 4-aminobutyrate) and lactate were monitored by using 13C-n.m.r. spectroscopy. When [1-13C]glucose was used as precursor the C-2 of 4-aminobutyrate was more highly labelled than the analogous C-4 of glutamate, whereas no label was observed in glutamine. A similar pattern was observed with [2-13C]glucose: the C-1 of 4-aminobutyrate was more highly labelled than the analogous C-5 of glutamate. Again, no labelling of glutamine was detected. In contrast, [2-13C]acetate labelled the C-4 of glutamine and the C-2 of 4-aminobutyrate more highly than the C-4 of glutamate; [1-13C]acetate also labelled the C-1 and C-5 positions of glutamine more than the analogous positions of glutamate. These results are consistent with earlier patterns reported from the use of 14C-labelled precursors that led to the concept of compartmentation of neuronal and glial metabolism and now provide the possibility of distinguishing differential effects of metabolic perturbations on the two pools simultaneously. An unexpected observation was that citrate is more highly labelled from acetate than from glucose.     

Substituent distribution on O,N-carboxymethylchitosans by 1H and 13C n.m.r.

The use of the n.m.r. method in the investigation of chitosan carboxymethylation was evaluated. It seems to be the most effective technique to determine concurrently the degree and the position of substitution of the carboxymethylated chitosan derivatives. The 13C-n.m.r., by the DEPT method, 1H-1H and 1H-13C-n.m.r. correlations give much valuable information from the chemical shifts of the complex carboxymethylchitosan spectra. The relative reactivity of the functional groups of chitosan towards carboxymethylation was also determined assuming a higher reactivity of the C-6 position.     

A two-dimensional 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) study of N-linked glycopeptides derived from calf fetuin

The oligosaccharide part of an N-linked triantennary glycopeptide from calf fetuin with fourteen carbohydrate residues and its smaller derivatives obtained by successive enzymic cleavage of the terminal residues were investigated using 2D 1H-n.m.r. (500 MHz) and 13C-n.m.r. (125 MHz) spectroscopy. Assignments have been made of the resonances of almost all the protons of the constituent carbohydrate residues in these glycopeptides. A comparison of the 1H chemical shifts and coupling constants, as determined from the cross-peaks, has shown the dependence of these parameters on the interactions of spatially related neighbouring carbohydrates. Small conformational changes take place upon elongation of the oligosaccharide side-chains.     

Structure and thermal interconversion of cyclobilirubin IX alpha.

One of the two main photoproducts in bilirubin metabolism during phototherapy in neonatal hyperbilirubinaemia is (EZ)-cyclobilirubin. However, it has not yet been possible to come to a final conclusion as to its chemical structure, despite the fact that much effort has been expended on the problem. The present paper demonstrates that (EZ)-cyclobilirubin is formed by the intramolecular cyclization of the C-3-vinyl group with the position at C-7 rather than at C-6, without delta-lactone-ring formation. The evidence comes from 13C-n.m.r. spectra, which indicate that an oxygen-bound quaternary carbon atom is not present, and from 1H-n.m.r. spectra, which indicate that the orientation of the methyl group at C-2 is equatorial; these findings are supported by mass spectra. The existence of both an epimeric relationship at C-7 between (EE)- and (EZ)-cyclobilirubins A and B and of steric isomers of the hydrogen atom and methyl group at C-2 is supported by the fact that the methyl-group protons at C-2 and C-7 are observed as a paired signal in 1H-n.m.r. spectra, and that new signals at C-7, C-2 and C-3 beta appear in 13C-n.m.r. spectra, that mass spectra of (EZ)-cyclobilirubins A and B are extremely similar and that, furthermore, thermal interconversion between (EE)- and (EZ)-cyclobilirubins A and B is observed.     

Isolation and identification of uridine(5'')-diphospho(1)-2,3-diacetamido-2,3-dideoxy-alpha-D- glucopyra nuronic acid from Pseudomonas aeruginosa P1-III.

A new uridine nucleotide was isolated from Pseudomonas aeruginosa P1-III (Habs serotype 5). On the basis of 13C-n.m.r. and p.m.r. spectroscopy, mass spectrometry, i.r.-absorption spectroscopy and circular dichrometry, the structure of the new compound was unequivocally identified as uridine(5')-diphospho(1)-2,3-diacetamido-2,3-dideoxy-alpha-D-gl ucopyranuronic acid.     

Analysis of bromine-oxidised dextran by 13C-n.m.r. spectroscopy

Dextran T 10, elaborated by Leuconostoc mesenteroides NRRL B-512, was oxidised with aqueous bromine at pH 7.0. The resulting oxodextran and its methoximated derivative were analysed by ¹³C-n.m.r. spectroscopy. The total amount of keto groups and their positions were established. Assignments of the ¹³C signals were made by referring to spectra of the corresponding methyl glucosiduloses and an oxodextran having most of the carbonyl groups at position 3 of the glycopyranosyl residues. In accordance with the mechanism for bromine oxidation of mono- and di-saccharides, the glucopyranosyl residues of dextran were oxidised mainly at C-2 and C-4. Over-oxidation resulted in a small proportion of acidic, ring-cleavage products.     

Use of 13C-n.m.r. spectroscopy for the quantitative estimation of 3-O- and 3,6-di-O-substituted D-glucopyranosyl residues in alpha-D-glucans formed by the D-glucosyltransferases of Streptococcus sobrinus

The 13C-n.m.r. spectra of the three alpha-D-glucans from Streptococcus sobrinus and the dextran from Leuconostoc mesenteroides, which differ widely in the ratios of omega (terminal, nonreducing) D-glucopyranosyl groups: 3-:6-:3,6-linked D-glucopyranosyl (Glc) residues, were measured in 0.5M NaOH at 22 degrees. The C-1 signals of 3-O-substituted Glc in a linear sequence, 6-O-substituted Glc in a linear sequence, 3,6-di-O-substituted Glc in a (1----6)-linked sequence, and Glc attached to O-3 of 3,6-di-O-substituted Glc were distinguished from each other. The C-3 signal of 3,6-linked Glc appeared downfield by 0.6 to 1.0 p.p.m. compared to the C-3 signal of 3-linked Glc in a linear sequence. The C-6 signals of omega-terminal, 3-linked, 6-linked, and 3,6-linked Glc were also assigned. The C-2 signal of 3-linked Glc in a linear sequence appeared separately, at 73.76 p.p.m. Based on these assignments, the various D-glucopyranosyl residues of the S. sobrinus alpha-D-glucans were quantitatively estimated from the signal areas of the C-2 atom of 3-linked Glc, the C-3 atom of 3-linked and 3,6-linked Glc, the C-6 atom of 6-linked and 3,6-linked Glc, and the C-6 atom of the omega-Glc groups and 3-linked Glc residues. The figures thus derived for the linkage ratios were close to those obtained by methylation analysis.     

13C-substituted pentos-2-uloses: synthesis and analysis by 1H- and 13C-n.m.r. spectroscopy

D-erythro-Pentos-2-ulose and D-threo-pentos-2-ulose and their 1-13C- and 2-13C-substituted derivatives have been prepared by oxidizing the corresponding natural and 13C-substituted D-aldopentoses (D-arabinose, D-xylose) with cupric acetate, and purifying the products by chromatography on a cation-exchange resin in the calcium or barium form. The equilibrium compositions of the pentos-2-uloses in 2H2O were determined by 13C-n.m.r. spectroscopy (75 MHz) at 25 degrees and 80 degrees. Among the eighteen possible monomeric acyclic, cyclic, and bicyclic forms, the anomeric pairs of the unhydrated aldopyranoses, aldopyranose endocyclic hydrates, aldofuranose endocyclic hydrates, and ketofuranose exocyclic hydrates were identified on the basis of 13C chemical shifts and 13C-1H and 13C-13C spin-coupling constants. 1H-N.m.r. (300, 500, and 620 MHz) and 13C-n.m.r. (75 MHz) spectroscopic data in one and two dimensions (DQF-COSY, homonuclear 2D-J) were used to evaluate the conformational properties of the cyclic structures. The unhydrated pyranoses are highly conformationally homogeneous; the erythro and threo isomers prefer 1C4 and 4C1 conformations, respectively. D-threo-Pentos-2-ulopyranose hydrate prefers the 4C1 conformation whereas the erythro isomers exists in both the 4C1 and 1C4 conformations. The furanoid forms favor structures having quasi-axial anomeric hydroxyl groups and quasi-equatorial exocyclic hydroxymethyl or dihydroxymethyl groups.     

Synthesis and n.m.r.-spectral analysis of unenriched and [1-13C]-enriched 5-deoxypentoses and 5-O-methylpentoses

Chemical methods are described for preparing unenriched and [1-13C]-enriched 5-deoxy- and 5-O-methyl-pentoses in the D or L configuration. The 1H-n.m.r. spectra of these compounds have been interpreted, and the 13C-n.m.r. spectra assigned with the aid of 2-D 13C-1H chemical-shift correlation spectroscopy. Tautomeric forms (furanoses, hydrate, and aldehyde) in solution in 2H2O have been quantified with the aid of [1-13C]-enriched derivatives. Spectra of 5-deoxypentoses, 5-O-methylpentoses, and methyl pentofuranosides have been compared, in order to assess the effect of 5-C-deoxygenation and 5-O-methylation on chemical shifts and coupling constants (1H-1H, 13C-1H, and 13C-13C) and on the pentofuranose conformations.     

A core oligosaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU843

The major oligosaccharide from the core region of the lipopolysaccharide from R. trifolii ANU843 was isolated and its structure determined. It is a trisaccharide consisting of two galacturonic acid residues and one 3-deoxy-D-manno-2-octulosonic acid (KDO) residue. The two galacturonic acid residues are terminally linked alpha to the C-4 and C-7 atoms of KDO. This structure was determined through use of 1H- and 13C-n.m.r. spectroscopy, f.a.b.-m.s., and g.l.c.-m.s. techniques. This oligosaccharide had not previously been reported to be present in the lipopolysaccharides from Gram-negative bacteria.     

Exopolysaccharides of the plant pathogens Pseudomonas corrugata and Ps. flavescens and the saprophyte Ps. chlororaphis

The rRNA-DNA homology group I pseudomonads Pseudomonas asplenii, Ps. corrugata, Ps. flavescens (plant pathogens), Ps. alcaligenes, Ps. pseudoalcaligenes subsp. pseudoalcaligenes (opportunistic human pathogens), Ps. aureofaciens and Ps. chlororaphis (saprophytes) were examined for their ability to produce exopolysaccharides (EPSs) when cultured on various solid and liquid complex media with glucose, glycerol or gluconate as primary sources of carbon. All three strains (388, 717 and ATCC 29736) of Ps. corrugata produced alginate, a polyuronan. An EPS composed of glucose, fucose, mannose and an unidentified uronic acid substituted with lactic acid was produced by one (B62) of two strains of Ps. flavescens. Of four strains of Ps. chlororaphis tested, only strain NRRL B-2075 produced EPS. The extracellular material purified by anion-exchange chromatography appeared to be a mixture of alginate plus an acidic hexosamine-containing polymer(s). Production of EPS by the other pseudomonads was not supported by any of the media tested.     

A 13C-N.M.R.-Spectral study of a gel-forming,branched (1→3)-β-d-glucan, (lentinan) from lentinus edodes, and its acid-degraded fractions. Structure,and dependence of conformation on the molecular weight

The structure and conformation of lentinan, an anti-tumor, branched (1→3)-β-d-glucan from Lentinus edodes, and its acid-degraded, lower molecular-weight fractions have been investigated by ¹³C-n.m.r. spectroscopy. It is found that their ¹³C-n.m.r. spectra are considerably changed, depending on the molecular weight. The conformational behavior as studied by ¹³C-n.m.r. spectroscopy is consistent with that revealed by a study of the shift in the absorption maximum of Congo Red complexed with lentinan and its acid-degraded fractions. It is found that the water-soluble fraction II (mol. wt. 3,640) gives rise to well-resolved ¹³C-n.m.r. spectra; the ¹³C-signals are assigned to (1→3)-β-d-glucan and branch points at C-6. The branched structure is also confirmed by examination of the ¹³C-n.m.r. spectra of the compounds in dimethyl sulfoxide. For the gel state of the fractions of higher molecular-weight, lentinan (mol. wt. 1,000,000) and fraction IV (mol. wt. 16,200), however, ¹³C-n.m.r. spectra of considerably attenuated signal-amplitude are observed. The fact that the ¹³C-signals of the β-d-(1→3)-linked main chain and side chains are completely suppressed is explained as a result of immobilization caused by their taking an ordered conformation. The ¹³C-resonances observed in the gel state, which are assigned to β-d-(1→6)-linkages, are unequivocally assigned to the side chains (of disordered conformation). Finally, the ordered conformation of both the β-d-(1→3)-linked main chain and side chains is identified as the single-helix conformation, which tends to form multiple helixes as junction zones for gel structure.     

4-O-(1-carboxyethyl)-D-galactose. A new acidic sugar from the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain 49.

The structure of a new acidic sugar from the extracellular polysaccharide of Butyrivibrio fibrisolvens strain 49 was determined as 4-O-(1-carboxyethyl)-D-galactose on the basis of 13C-n.m.r. and 1H-n.m.r. spectroscopy, m.s. and chemical degradation studies.