An o-toluidine method for detection of carbohydrates in protein hydrolysates

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.     

High-performance liquid chromatography of monosaccharides and oligosaccharides in a complex biological matrix.

Analysis of oligosaccharides in complex biological matrices is hampered by the fact that oligosaccharides, closely related in structure, are difficult to separate from each other and that conventional detection procedures (refraction index and uv detection) are not specific enough for carbohydrates. Prepurification of samples by procedures like desalting or gel filtration is often used but can lead to the loss of specific oligosaccharides. We have used pellicular anion chromatography in combination with a postcolumn reaction for reducing carbohydrates based on 4-aminobenzoylhydrazide. This procedure not only detected normal mono- and oligosaccharides but N-acetylhexosamines and reducing N-acetylhexosamine containing oligosaccharides as well. A sensitivity of about 20-25 pmol for non-GlcNAc containing mono- or oligosaccharides and between 30-50 pmol for GlcNAc or oligosaccharides with GlcNAc at the reducing side was reached. The postcolumn detection was compared with pulsed amperometric detection and appeared to be more specific for mono- and oligosaccharides. Except for deproteination to protect the column, no further sample preparation was needed with this system for our application (urines). In this way pellicular anion chromatography in combination with this postcolumn reaction reaction to be a sensitive and specific HPLC procedure for analysis of monosaccharides and oligosaccharides in complex biological matrices.     

Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection

Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

A high performance liquid chromatography method for the analysis of glycosphingolipids using galactose oxidase/NaB3H4 labeling of intact cells and synaptosomes

The major objective of this study was to combine an HPLC method with a galactose oxidase/NaB3H4 labeling method to allow both a chemical quantitation of individual glycolipids and analysis of their 3H labeling. Neutral glycolipids in whole cells were oxidized with galactose oxidase, and the resultant aldehydes were radiolabeled by reduction with tritiated sodium borohydride. Gangliosides, oxidized with galactose oxidase, either were reduced while in the native state in the whole cell or were first extracted and then reduced. Tritiated glycolipids were perbenzoylated and separated by HPLC. Ultraviolet detection of the derivatives was at 230 nm. Incorporated radioactivity was determined either by collecting fractions from the HPLC separation and counting on a liquid scintillation spectrometer or with a flow-through counter. The order of the derivatization and reduction is critical. Reduction of glycolipids prior to derivatization yielded sharp uv and radioactive peaks. Perbenzoylation of the oxidized glycolipids prior to reduction yielded multiple uv peaks, a noisy baseline, and broad radioactive peaks which did not always have a corresponding uv peak. The labeled neutral glycolipids were stable at -40 degrees C for at least 14 days, and gangliosides were stable at -15 degrees C for at least 14 days. When samples were stored at 20 degrees C there was a time-dependent decrease in the glycolipid/internal standard uv peak area ratio for GbOse4 and GbOse3 apparent by 28 days after perbenzoylation. The distribution of radiolabel among peaks showed no change with time or temperature. We adapted the technique to allow 3H labeling of glycolipids from monolayers of cultured glioma cells and from mouse brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial surface display of GFP(uv) on bacillus subtilis spores

To analyze a cotG-based Bacillus subtilis spore display system directly, GFP(uv) was expressed on the surface of Bacillus subtilis spores. When GFP(uv) was fused to the C-terminal of the cotG structural gene and expressed, the existence of a CotG-GFP(uv) fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This GFP(uv) displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.     

Femtomole oligosaccharide detection using a reducing-end derivative and chemical ionization mass spectrometry

A bimodal reagent (pentafluorobenzyl aminobenzoate) has been synthesized to improve oligosaccharide isolation, detection, and structural characterization. The reagent is glycosidically attached to the reducing end of glycan residues, imparts fluorescent and uv properties for chromatographic detection, and functions as an efficient electron trap under negative ion chemical ionization mass spectrometry for femtomole detectability. Facile ester cleavage and pentafluorobenzyl elimination provides a single molecular-weight-related fragment in high abundance. Procedures are described for reagent synthesis, purification, and oligosaccharide conjugation. Carbohydrate samples derivatized with this reagent are evaluated by high-performance liquid chromatography and supercritical fluid chromatography (SFC) and for sensitivity by SFC negative ion chemical ionization mass spectrometry.     

Detection of prostaglandins by high-performance liquid chromatography after conversion to p-(9-anthroyloxy)phenacyl esters

p-(9-Anthroyloxy)phenacyl bromide (panacyl bromide) undergoes rapid reaction with the carboxyl group of prostaglandins in the presence of N,N-diisopropylethylamine in acetonitrile-tetrahydrofuran (4:1). The resulting prostaglandin panacyl esters are strongly uv absorbing with a lambda max at 253 nm and an epsilon of 174,280 in acetonitrile. The lower limit of detection of prostaglandins was approximately 200 pg with uv detection (254 nm) and about 30 pg with fluorescent detection (exitation 253 and emission 445 nm) using normal-phase HPLC. The reactivity of panacyl bromide with 23 prostaglandins as well as prostaglandins released by human lung tissues was investigated.     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

A new method for pH determination in isoelectric focusing experiments

A rapid and reproducible method for the pH measurement in the effluent from density gradient electrofocusing is described. By this procedure, after preparative isoelectric focusing, the detection of protein zones and pH measurement can be accomplished simultaneously, by serially coupling a uv flow cell with a pH flow cell. This last one is connected to the recorder by a control unit, which allows the simultaneous printing of pH and uv absorption on the same chart.     

Amino and hydrazino alkyl benzoates as derivatizing agents for the separation and mass spectrometric analysis of oligosaccharides from bacterial lipooligosaccharides

In an attempt to develop more sensitive and versatile methods for the structure analysis of oligosaccharides derived from lipooligosaccharides (LOS) of gram-negative bacteria, amino and hydrazino alkyl benzoate derivatives were prepared. These oligosaccharide derivatives were separated by HPLC and then analyzed by liquid secondary ion mass spectrometry (LSIMS). Both the amino and hydrazino alkyl benzoates react with the free reducing termini of acid-treated LOS, increasing the hydrophobicity of the released oligosaccharides and allowing them to be separated by reverse-phase HPLC. In addition, these oligosaccharide derivatives now contain a sensitive uv chromophore for subsequent peak detection and improve the quality of the LSIMS spectra compared to underivatized oligosaccharides. However, the amino alkyl benzoates reacted poorly compared to the analogous hydrazino alkyl benzoates with 3-deoxy-manno-2-keto octulosonic acid (KDO), and oligosaccharides with KDO at the reducing terminus, especially when the oligosaccharide also contained phosphoethanolamine. Derivatization with the hydrazino compounds can be carried out quickly and under mild conditions using a minimal amount of reagent, and is therefore suitable for microscale analyses. The chromatographic and mass spectrometric characteristics of these derivatives make them excellent alternatives to permethylation and peracetylation techniques for the structural analysis of complex bacterial oligosaccharides derived from glycolipids.     

Ultraviolet nicking of large DNA molecules from pulsed-field gels for southern transfer and hybridization.

Large DNA molecules separated in pulsed-field gels are not efficiently transferred from the gel for Southern hybridization. Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported. We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detection by hybridization. To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered. Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis.     

Selective T cell killing of human lymphocytes by ultraviolet radiation

The effects of ultraviolet radiation (uv) on human B and T lymphocytes were studied. In vitro studies showed that T lymphocytes were more sensitive to uv than B lymphocytes as assessed by eosin-dye exclusion. Following uv exposure, the viable lymphocytes responded to mitogens (PHA, PWM), and functional B lymphocytes were present at a time when no viable T cells were detected. Varying doses of uv were required to abrogate different in vitro responses (proliferative response to antigen or allogeneic cells, MIF production, and cell-mediated lympholysis). In vivo, uv was able to diminish an established cutaneous delayed hypersensitivity response. In vitro uv treatment of parental mouse spleen cells eliminated a graft-versus-host reaction in F₁ recipients as determined by the spleen index. The basis for the differential effect of uv on B and T lymphocyte viability and functional responses is unknown.     

Determination of alpha-amanitin and beta-amanitin in human biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic assay of alpha-amanitin and beta-amanitin in human serum, urine, or stomach washings is described. Sample preparation involves a chemical step with deproteinization and organic solvent treatment, and a selective cleanup and concentration step on reversed-phase prepacked cartridges. Separations are performed on a reversed-phase analytical column under isocratic conditions with uv detection at 280 nm. The method allows the quantitation of alpha- and beta-amanitin separately with a detection limit of 10 ng/ml for both toxins.     

High-performance liquid chromatographic analysis of fucoganglioside hydrolysis by alpha-L-fucosidase

A sensitive HPLC method has been developed for monitoring fucoganglioside hydrolysis by purified alpha-L-fucosidase. The high-resolution method employs a Lichrosorb-NH2 column, a 10-min isocratic elution with potassium phosphate/acetonitrile buffer, detection of ganglioside products with a uv monitor at 195 nm, and quantification of low picomolar amounts of these gangliosides with an integrator. The usefulness of the HPLC method has been exemplified by using it to demonstrate the hydrolysis of gangliosides fucosyl-GM1 and fucosyl-GD1b by purified human liver alpha-L-fucosidase in the absence of activator proteins and/or detergents.     

Quantitation of ultraviolet radiation-induced cyclobutyl pyrimidine dimers in DNA by video and photographic densitometry

We have compared video and photographic methods for calculating the number of ultraviolet radiation (uv)-induced pyrimidine dimers in DNA from the bacteriophage T7 exposed to uv (0 to 800 J/m2) from an FS40 sunlamp. DNA was incubated with a pyrimidine dimer-specific Micrococcus luteus uv endonuclease, subjected to alkaline agarose gel electrophoresis, neutralized, and stained with ethidium bromide, and the DNA fluorescence was recorded either with a video camera or on photographic film. The slopes of the dose-response curves for the number of uv-endonuclease-sensitive sites per 10(3) bases (pyrimidine dimers) was 1.2 (+/- 0.1) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the video analysis and 1.3 (+/- 0.04) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the photographic analysis. Results for pyrimidine dimer determination by either method were statistically comparable.     

Ultraviolet Rays Induced Expression of Lectins on the Surface of a Squamous Carcinoma Keratinocyte Cell Line

Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either α-l-rhamnosyl or α-d-glucosyl residues. A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sun-like doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells. The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation. Such a specific modulation of receptor expression upon uv irradiation might be biologically significant, considering the numerous intercellular recognition phenomena in skin biology. α-l-Rhamnose-specific receptor on SCL-1 could not be distinguished from α-d-glucose-specific receptor on the basis of neoglycoproteins binding, uptake, and related inhibitions. Lectin expression was mainly detected on the cell surface, and its overexpression due to uv rays required ade novoprotein synthesis process.     

Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.     

Simultaneous determination of tocopherols,ubiquinols, and ubiquinones in blood,plasma, tissue homogenates,and subcellular fractions

A fast single-step lipid extraction procedure and high-performance liquid chromatography with in-line uv and electrochemical detection are used for the simultaneous quantitative determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. The compounds of interest can be quantitatively extracted into hexane from a sodium dodecyl sulfate-treated aqueous homogenate after precipitation of protein by addition of an equal volume of ethanol. α-, γ-, and δ-Tocopherol, ubiquinol 9, ubiquinol 10, and ubiquinones 9 and 10 can be well separated on a reversed phase column. Ubiquinones are detected at 275 nm by the uv detector, and ubiquinols and tocopherols by the electrochemical detector in the oxidative mode. Quantitation is done by comparing chromatographic peak heights to those of a standard solution containing known amounts of tocopherols, ubiquinols 9 and 10, and ubiquinones 9 and 10, analyzed under identical conditions. The high sensitivity of the electrochemical detection allows operation at low potentials (+0.5 V) with low detector response, but high selectivity for the easily oxidizable tocopherols and ubiquinols and decreased baseline noise. The uv detection limits the overall sensitivity of the procedure to 2 pmol ubiquinone, corresponding to 0.1 μm ubiquinone in the lipid extract. The ranges of values obtained for rat and guinea pig tissues, for rat liver mitochondria, and for blood and plasma from rats and humans are given.     

Selective quantification of arachidonic acid hydroperoxides and their hydroxy derivatives in reverse-phase high performance liquid chromatography

For the quantification of lipid hydroperoxides by high performance liquid chromatography (HPLC), it has been necessary to improve the detection system specific to the hydroperoxy group. We first developed a technique which combined detection by uv absorption due to conjugated diene and detection based on electrochemical (EC) reduction in reverse-phase HPLC for the selective determination of arachidonic acid hydroperoxides (hydroperoxyeicosatetraenoic acid, HPETE) and its reduced derivative, hydroxyeicosatetraenoic acid (HETE). 15-HPETE was quantified selectively by EC detection, although both 15-HPETE and 15-HETE were detected by uv absorption and were hardly resolved in the chromatogram. Isomers in HPETE obtained from autoxidized arachidonic acid were partially separated in the chromatogram and seem to have been quantified similarly to 15-HPETE. The application of this analytical system to the analysis of 15-HPETE added in human plasma has demonstrated that the recovery of HPETE extracted from human plasma is much lower than that from normal saline and that HPETE is reduced to HETE by incubation at 37 degrees C. The fact that a high concentration of glutathione accelerated this reduction may indicate that human plasma possesses a glutathione-dependent HPETE-reducing ability as a defense system against excess accumulation of lipid hydroperoxides. Blood plasma effectively suppressed the decomposition of HPETE induced by ferrous ion indicating the presence of factors which prevent the action of ferrous ion on HPETE.