An enzyme-linked immunosorbent assay for the detection of diacetyl (2,3-butanedione).

Diacetyl (2,3-butanedione) is an important metabolic marker of several cancers, as well as an important off-flavour component produced during fermentation. As a small molecule in a complex mixture with many other analytes, existing methods for identification and quantitation of diacetyl invariably involves a chromatographic separation step followed by signal integration with an appropriate stoichiometric detector. Here we demonstrate that the chemical reaction of diacetyl with a 1,2-phenylenediamine derivative yields a chemical adduct, 1,4-quinoxaline which can be conjugated on BSA. The BSA-diacetyl adduct can be used to select an adduct-specific monoclonal antibody in a Fab-format from a 45-billion member phage-display library. The availability of this antibody allowed the development of an enzyme-linked immunosorbent assay for diacetyl, based on the 1,4-quinoxaline competition for the antibodies with the diacetyl adduct immobilized on the plate. The described ELISA assay can detect the captured diacetyl in micromolar concentrations, both in water samples and in cell culture medium.

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley ß-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.

A chromogenic assay for limit dextrinase and pullulanase activity.

A new chromogenic substrate to assay the starch debranching enzymes limit dextrinase and pullulanase is described. The 2-chloro-4-nitrophenyl glycoside of a commercially available branched heptasaccharide (Glc-maltotriosyl-maltotriose) was found to be a suitable specific substrate for starch debranching enzymes and allows convenient assays of enzymatic activities in a format suited for high-throughput analysis. The kinetic parameters of these enzymes toward the synthesized substrate are determined, and the selectivity of the substrate in a complex cereal-based extract is established.

Profiling of carbohydrate mixtures at unprecedented resolution using high-precision 1H-13C chemical shift measurements and a reference library.

Complex mixtures of carbohydrates pose distinct challenges in routine and high-throughput analysis, so that only a few carbohydrate components are routinely resolved and identified in biofluids, extracts, foods and other complex mixtures. Here, we conduct precise measurements of (1)H and (13)C anomeric chemical shifts to construct a reference library of specific carbohydrate signals with high-resolution two-dimensional (1)H-(13)C NMR spectra. High-resolution multidimensional NMR spectra largely abolish resolution problems in carbohydrate analysis with state-of-the-art instrumentation. Accurate measurements of anomeric (1)H-(13)C chemical shifts at parts per billion precisions permit robust carbohydrate identification using a very limited number of instrument-independent reference values.

Fast and accurate quantitation of glucans in complex mixtures by optimized heteronuclear NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a widely used technique for mixture analysis, but it has shortcomings in resolving carbohydrate mixtures due to the narrow chemical shift range of glycans in general and fragments of homopolymers in particular. Here, we suggest a protocol toward fast spectroscopic glycan mixture analysis. We show that a plethora of oligosaccharides comprising only α-glucopyranosyl residues can be resolved into distinct quantifiable signals with NMR experiments that are substantially faster than chromatographic runs. Conceptually, the approach fully exploits the narrow line widths of glycans (ν1/2 < 3 Hz) in the (13)C spectral dimension while disregarding superfluous spectral information in compound identification and quantitation. The acetal (H1C1) groups suffice to spectroscopically resolve ∼20 different starch fragments in optimized (1)H-(13)C NMR with a narrow (13)C spectral width (3 ppm) that allows sampling the indirect (13)C dimension at high resolution within 15 min. Rapid quantitations by high-resolution NMR data are achieved for glycans at concentrations as low as 10 µg/mL. For validation, comparisons were made with quantitations obtained by more time-consuming chromatographic methods and yielded coefficients of determination (R(2)) above 0.99.

Time-resolved in-situ observation of starch polysaccharide degradation pathways.

Analytical challenges in the direct time-resolved observation of starch metabolism have been addressed by using optimized multidimensional NMR experiments. Starch provides the main source of human dietary energy intake and is a raw material for beverage and renewable fuel production. Use of direct in situ observations of starch remodeling pathways could facilitate our understanding and control of processes of biotechnological, medical, and environmental relevance. Processes involving starch synthesis or degradation are difficult to monitor directly in aqueous solution, however, because starch consists of glucopyranosyl homopolymers that are built up from and degraded into structurally similar fragments that yield only small signal dispersion in optical and NMR spectroscopy. By focusing on acetal groups only, (1) H,(13) C HSQC experiments sampling narrow spectral windows in the highly resolved (13) C dimension have been employed in order to observe the amylopectin cleavage pathway in real time with a temporal resolution of 150 s. Quantifiable signals for more than 15 molecular species emerging during starch fragmentation by human saliva have been resolved and tracked over time in this manner. Altered accumulation of intermediates in the digestion of amylopectin in the presence of black tea acting as an effector have been monitored.

Probing helical hydrophobic binding sites in branched starch polysaccharides using NMR spectroscopy.

Branched starch polysaccharides are capable of binding multiple hydrophobic guests, but their exploitation as multivalent hosts and in functional materials is limited by their structural complexity and diversity. Linear α(1-4)-linked glucose oligosaccharides are known to bind hydrophobic guests inside left-handed single helices in solution and the solid state. Here, we describe the development of an amphiphilic probe that binds to linear α(1-4)-linked glucose oligosaccharides and undergoes a conformational switch upon complexation, which gives rise to dramatic changes in the (1)H NMR spectrum of the probe. We use this probe to explore hydrophobic binding sites in the branched starch polysaccharides amylopectin and ß-limit dextrin. Diffusion-ordered (DOSY), nuclear Overhauser effect (NOESY) and chemical shift perturbation (HSQC) NMR experiments are utilised to provide evidence that, in aqueous solution, branched polysaccharides bind hydrophobic guests in well-defined helical binding sites, similar to those reported for complexation by linear oligosaccharides. By examining the binding affinity of the probe to systematically enzymatically degraded polysaccharides, we deduce that the binding sites for hydrophobic guests can be located on internal as well as external branches and that proximal α(1-6)-linked branch points weaken but do not prevent complexation.

Capillary electrophoresis with three-color fluorescence detection for the analysis of glycosphingolipid metabolism.

A capillary electrophoresis system with an ultrasensitive three-color laser-induced fluorescence detector was constructed for the simultaneous measurement of glycosphingolipids conjugated with a family of BODIPY fluorophores. The compounds were separated by capillary electrophoresis and detected by laser-induced fluorescence excited within a sheath-flow cuvette. Diode-pumped solid-state lasers operating at 473 nm and 532 nm, and a diode laser operating at 633 nm were used to excite glycosphingolipids tagged with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 fluorophores. Detection limits were 34 ± 1 molecules, 67 ± 7 molecules, and 36 ± 13 molecules of BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled glycosphingolipids. Separation efficiencies were typically one million theoretical plates. To test the ability of the system to analyze cellular contents in an in vitro biological model, differentiated PC12 cells were co-incubated with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled lactosylceramide substrates. Cells were homogenized. The metabolic products originating from the glycosphingolipid substrates were simultaneously analyzed using the system.

A convenient synthesis of GDP-D-rhamnose: the donor substrate for D-rhamnosyltransferase WbpZ from Pseudomonas aeruginosa PAO1.

Gram negative bacteria have lipopolysaccharides (LPS) that are critical for their survival. LPS molecules are composed of antigenic exopolysaccharide chains (O antigens). We are interested in discovering the enzymes involved in the biosynthesis of O antigens in Pseudomonas aeruginosa. The common polysaccharide antigen contains α-linked D-rhamnose residues. We have now synthesized GDP-D-rhamnose by a convenient synthesis in aqueous solution, and have shown that it can be used without extensive purification as the donor substrate for D-rhamnosyltransferase (WbpZ) from the P. aeruginosa strain PAO1. The availability of this nucleotide sugar preparation allows for characterization of D-rhamnosyltransferases.

Nature's dendrimer: characterizing amylopectin as a multivalent host.

Hang on to those branches! Amylopectin, the major polysaccharide of starch, is a predominantly α(1,4)-linked glucan whose properties are defined by its size and the number, distribution, and length of its α(1,6)-linked branches. The amphiphilic probe HPTS-C16 H33 binds to terminal helical branches longer than 12 glucose units (green), which allows for a detailed quantitative characterization of polysaccharide branching by (1) H NMR spectroscopy.

Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates.

Mucin-type glycosylation [α-N-acetyl-D-galactosamine (α-GalNAc)-O-Ser/Thr] on proteins is initiated biosynthetically by 16 homologous isoforms of GalNAc-Ts (uridine diphosphate-GalNAc:polypeptide N-acetylgalactosaminyltransferases). All the GalNAc-Ts consist of a catalytic domain and a lectin domain. Previous reports of GalNAc-T assays toward peptides and α-GalNAc glycopeptides showed that the lectin domain recognized the sugar on the substrates and affected the reaction; however, the details are not clear. Here, we report a new strategy to give insight on the sugar recognition ability and the function of the GalNAc-T3 lectin domain using chemically synthesized natural-type (α-GalNAc-O-Thr) and unnatural-type [ß-GalNAc-O-Thr, α-Fuc-O-Thr and ß-GlcNAc-O-Thr] MUC5AC glycopeptides. GalNAc-T3 is one of isoforms expressed in various organs, its substrate specificity extensively characterized and its anomalous expression has been identified in several types of cancer (e.g. pancreas and stomach). The glycopeptides used in this study were designed based on a preliminary peptide assay with a sequence derived from the MUC5AC tandem repeat. Through GalNAc-T3 and lectin-inactivated GalNAc-T3, competition assays between the glycopeptide substrates and product analyses (MALDI-TOF MS, RP-HPLC and ETD-MS/MS), we show that the lectin domain strictly recognized GalNAc on the substrate and this specificity controlled the glycosylation pathway.

Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons.

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

Thiogalactopyranosides are resistant to hydrolysis by α-galactosidases.

Fluorescently tagged glycosides containing terminal α(1→3) and α(1→4)-linked thiogalactopyranosides have been prepared and tested for resistance to hydrolysis by α-galactosidases. Eight fluorescent glycosides containing either galactose or 5-thiogalactose as the terminal sugar were enzymatically synthesized using galactosyltransferases, with lactosyl glycosides as acceptors and UDP-galactose or UDP-5'-thiogalactose, respectively, as donors. The glycosides were incubated with human α-galactosidase A (CAZy family GH27, a retaining glycosidase), Bacteroides fragilis α-1,3-galactosidase (GH110, an inverting glycosidase), or homogenates of MCF-7 human breast cancer cells or NG108-15 rat glioma cells. Substrate hydrolysis was monitored by capillary electrophoresis with fluorescence detection. All compounds containing terminal O-galactose were readily degraded. Their 5-thiogalactose counterparts were resistant to hydrolysis by human α-galactosidase A and the enzymes present in the cell extracts. B. fragilis α-1,3-galactosidase hydrolyzed both thio- and O-galactoside substrates; however, the thiogalactosides were hydrolyzed at only 1-3 % of the rate of O-galactosides. The hydrolytic resistance of 5-thiogalactose was also confirmed by an in vivo study using cells in culture. The results suggest that 5-thiogalactosides may be useful tools for the study of anabolic pathways in cell extracts or in single cells.

Single cell ganglioside catabolism in primary cerebellar neurons and glia.

Cell-to-cell heterogeneity in ganglioside catabolism was determined by profiling fluorescent tetramethylrhodamine-labeled GM1 (TMR-GM1) breakdown in individual primary neurons and glia from the rat cerebellum. Cells isolated from 5 to 6 day old rat cerebella were cultured for 7 days, and then incubated for 14 h with TMR-GM1. Intact cells were recovered from cultures by mild proteolysis, paraformaldehyde fixed, and subjected to single cell analysis. Individual cells were captured in a capillary, lysed, and the released single-cell contents analyzed by capillary electrophoresis with quantitative laser-induced fluorescent detection of metabolites. Non-neuronal cells on average took up much more exogenous TMR-GM1 than neuronal cells, and catabolized it more extensively. After 14 h of incubation, non-neuronal cells retained only 14% of the TMR products as GM1 and GM2, compared to >50% for neurons. On average, non-neuronal cells contained 74% of TMR-labeled product as TMR-ceramide, compared to only 42% for neurons. Non-neuronal cells retained seven times as much TMR-GM3 (7%) compared to neuronal cells (1%). To confirm the observed single cell metabolomics, we lysed and compared TMR-GM1 catabolic profiles from mixed neuron/glial cell cultures and from cultures depleted of non-neuronal cells by treatment with the antimitotic agent cytosine arabinoside. The lysed culture catabolic profiles were consistent with the average profiles of single neurons and glia. We conclude that the ultrasensitive analytic methods described accurately reflect single cell ganglioside catabolism in different cell populations from the brain.

Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.

Probing the acceptor substrate binding site of Trypanosoma cruzi trans-sialidase with systematically modified substrates and glycoside libraries.

Systematically modified octyl galactosides and octyl N-acetyllactosamines were assessed as inhibitors of, and substrates for, T. cruzi trans-sialidase (TcTS) in the context of exploring its acceptor substrate binding site. These studies show that TcTS, which catalyses the α-(2→3)-sialylation of non-reducing terminal ß-galactose residues, is largely intolerant of substitution of the galactose 2 and 4 positions whereas substitution of the galactose 6 position is well tolerated. Further studies show that even the addition of a bulky sugar residue (glucose, galactose) does not impact negatively on TcTS binding and turnover, which highlights the potential of 'internal' 6-substituted galactose residues to serve as TcTS acceptor substrates. Results from screening a 93-membered thiogalactoside library highlight a number of structural features (notably imidazoles and indoles) that are worthy of further investigation in the context of TcTS inhibitor development.

Monitoring the uptake of glycosphingolipids in Plasmodium falciparum-infected erythrocytes using both fluorescence microscopy and capillary electrophoresis with laser-induced fluorescence detection.

The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique.

Nucleophilic catalysis of carbohydrate oxime formation by anilines.

Chemoselective formation of glycoconjugates from unprotected glycans is needed to further develop chemical biology involving glycans. Carbohydrate oxime formation is often slow, and organocatalysis by anilines would be highly promising. Here, we present that carbohydrate oxime formation can be catalyzed with up to 20-fold increases in overall reaction rate at 100 mM aniline. Application of this methodology provided access to complex glycoconjugates.

The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells.

E-, P- and L-selectins critically function in lymphocyte recirculation and recruiting leukocytes to inflammatory sites. MECA-79 antibody inhibits L-selectin-mediated lymphocyte adhesion in several species and does not require sialic acid in its epitope. Many other antibodies, however, recognize human selectin ligands expressing N-acetylneuraminic acid but not mouse selectin ligands expressing N-glycolylneuraminic acid, suggesting that difference in sialic acid in sialyl Lewis X leads to differential reactivity. We found that HECA-452 and FH6 monoclonal antibodies bind Chinese hamster ovary (CHO) cells expressing N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl form. Moreover, synthetic N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl oligosaccharide inhibited HECA-452 and FH6 binding. By contrast, E-, P- and L-selectin bound to CHO cells regardless of whether they express N-acetyl or N-glycolyl form of sialyl Lewis X, showing that selectins have a broader recognition capacity than HECA-452 and FH-6 anti-sialyl Lewis x antibodies.