The rapid,quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas-liquid chromatography

O-Methyloximes have been prepared from 2-amino-2-deoxy-d-glucose, -d-mannose, and -d-galactose. The acetates of these derivatives yield stable compounds which are readily separated quantitatively by gas-liquid chromatography on a number of polar phases. The above compounds along with the aldononitrile acetates of neutral sugars can be easily separated from one another on a single column in one chromatographic run. The procedures developed were tested on a number of glycoproteins of known composition as reported by other workers utilizing more classical methodologies, resulting in excellent agreement in terms of sugar composition. An improved method is also described for converting neutral sugars to oximes which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

Amino acid analysis by gas-liquid chromatography on a single column

A gas-liquid chromatography procedure for analysis of protein amino acids is described. Amino acids are esterified to their n-propyl esters then acylated to their heptafluorobutyryl (HFB) derivatives. These reactions were carried out in a single tube at 100°C. A simple steam-heating apparatus was constructed that heats only the bottom of the reaction vessel. Only 10 min were needed for esterification and 20 min for acylation, respectively. The resulting products, N-HFB-n-propyl esters of amino acids, were chromatographed on a single column. The amino acid compositions of chymotrypsinogen A and casein were analyzed by the present method, and the results were compared with those obtained by ion-exchange chromatography reported previously.     

Estimation of disaccharides in plasma and urine by gas-liquid chromatography

A technique for the estimation of disaccharides in plasma and urine using gas—liquid chromatography is described. The procedure involves the formation of trimethylsilyl derivatives followed by injection of the reaction mixture directly onto the column. The method is precise, linear over a wide range and gives recoveries of 93—99%. The limit of sensitivity is 80 μg per 100 ml, but with modification of the volumes used, levels of 40 μg per 100 ml may be quantitated.     

Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography

All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH₂-terminal analysis of lysozyme is described.     

Quantitative gas-liquid chromatographic determination of ftorafur and 5-fluorouracil in biological specimens

A method developed for measuring the antitumor agent ftorafur and its biotransformation product 5-fluorouracil was applied to biological specimens. After extraction with ethylacetate, ftorafur, 5-fluorouracil, and the internal standard 2-methyl-4-hydroxy-6-chloromethylpyrimidine are converted to their chloromethyldimethylsilyl derivatives and assayed by glc, using either an electron-capture or a flame ionization detector. The minimum detectable amount is 100 pg/injection for ftorafur and 50 pg/injection for 5-fluorouracil employing electron-capture detection. Linearity was found up to microgram amounts of both substances, without any interference from endogenous substrates. Preliminary data are reported on the comparative serum kinetics of ftorafur and 5-fluorouracil in mice.     

Quantitative determination of partially methylated alditol acetate of amino sugar by gas chromatography-mass spectrometry

Using doubly labeled N-acetyllactosamine. Hakomori's procedures to prepare partially methylated alditol acetates and their subsequent analysis by gas chromatography-mass spectrometry were followed from a quantitative aspect. It was found that both galactose and glucosamine were nearly quantitatively converted to PMAAs. Preferential loss of PMAA of glucosamine occurred on a column for gas chromatography when the amount of the PMAA injected onto a column was less than a certain level. Above this level, PMAAs of both galactose and glucosamine were eluted from the column with equal yields. However, in mass spectrometry with monitoring of total ions, the molar response factor of PMAA of glucosamine was found to be about 25% higher than that of PMAA of galactose. Based on these findings, methylation analysis of an oligosaccharide from Taka-amylase A composed of one glucosamine and five mannose residues could be carried out quantitatively.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Separation and identification of O-acetyl-O-methyl-galactononitriles by gas-liquid chromatography and mass spectrometry

The gas-liquid-chromatographic retention-times and the mass-spectral features of partially methylated d-galactononitrile acetates are reported. Distinctive fragmentation of each of the mono-O-methyl derivatives allows their identification, and the results are applicable to the same substituted derivatives of the other aldohexoses. A new fragmentation-pathway, typical of the acetylated and the O-acetyl-O-methylaldononitriles, is proposed in order to justify previously unexplained fragments. This fragmentation competes with the known ones in derivatives that do not carry vicinal methoxyl groups.     

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection

We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.     

A quantitative study of organic phosphate-amine interaction by 31P nuclear magnetic resonance

Interactions between the phosphate group of 4-deoxypyridoxine 5′-phosphate and different protonated amines were quantitatively measured by means of {³¹P}-¹H nuclear magnetic double resonance technique combined with pD titration. An interaction of the phosphate group with added amine resulted in a measurable difference in the ³¹P chemical shift of these phosphate-containing samples with and without amine [Δδ(³¹P)]. Basic amino acids and biogenic amines had significant measurable Δδ(³¹P) values. No interactions were observed for acidic or neutral α, β and γ-amino acids.     

A sensitive assay of galactocerebroside beta-galactosidase by high-performance liquid chromatography using galactosyl-N-dansyl-sphingosine as substrate

A rapid and sensitive method was devised for determining β-galactosidase activity specific for galactocerebroside. A fluorescent derivative of galactocerebroside, 1-O-galactosyl-2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was used as substrate, and the product, 2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was taken into organic solvent phase. Quantitative analysis of 2-N-dimethylaminonaphthalene-5-sulfonyl-sphingosine was carried out fluorometrically by use of high-performance liquid chromatography on silica gel column.     

A rat kidney neutral peptidase that degrades B chain of insulin, glucagon, and ACTH: purification by affinity chromatography and some properties.

A metallo-endopeptidase that catalyzes at near neutral pH the hydrolysis of certain polypeptides was purified from rat kidney microsomes by a simplified procedure using affinity chromatography on Sepharose 4B coupled with insulin B chain. The purified enzyme showed a single component by chromatography on diethylaminoethyl cellulose and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 213,000. Studies on its substrate specificity showed that the purified enzyme rapidly degrades insulin B chain, glucagon, adrenocorticotropin, and, at a significantly lower rate, insulin A chain. The enzyme has a very weak or no activity toward ribonuclease and vasopressin. In contrast, the enzyme does not degrade denatured hemoglobin, bovine serum albumin, insulin (nano- or micromolar), oxytocin, furylacryloylglycyl-leucine amide (FAGLA), synthetic substrates of cathepsin C (β-napthalamides of glycine-l-arginine and l-histidine-l-serine), or synthetic substrates of aminopeptidases (l-arginine- or l-glutamic acid-β-napthylamide). The enzyme degrades reduced or oxidized B chain at about the same rate, but S-sulfonated B chain is degraded at a markedly lower rate. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. Activity of the enzyme is markedly inhibited by chelating agents (EDTA and o-phenanthroline) and, modestly, by high concentrations of citrate and histidine. Activity of the enzyme is also markedly inhibited by simple thiol compounds (dithiothreitol, glutathione, and mercaptoethanol), but not by sulfhydryl reagents (N-ethylmaleimide or iodoacetate). The inactive apoenzyme, prepared by treatment of the enzyme with EDTA followed by dialysis, was reactivated by Zn²⁺ > Ca²⁺, minimally by Cu²⁺, but not by Hg²⁺. Some anions (phosphate, borate, and bicarbonate) were strongly inhibitory, but chloride had no effect. The following agents were found to have no effect: soybean and lima bean trypsin inhibitors, N^?-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N^α,?-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin (Trasylol), phenylmethylsulfonyl fluoride (a serine protease inhibitor), 1-methyl histidine, 3-methyl histidine, histamine, imidazole, and heparin.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

A quantitative determination of the relative amount of histones in polyacrylamide gel by silver stain

On the basis of scanning densitometry of the stained gel, the conditions for the quantitative determination of individual histones by silver was examined and compared with the dye-staining method, in terms of higher sensitivity and faithful quantitation. Fixation with formaldehyde, coupled with simultaneous prestaining with Coomassie brilliant blue (CBB), was found to be most suitable. Prior fixation in acidic alcohol alone failed to stain the histones accurately, but this failure could be partly alleviated by prestaining with CBB. Although the sensitivity for detecting histones by silver staining is lower than that for neutral proteins by about 10-fold, it is at least 10-fold higher than the CBB stain.     

Simple,rapid, and highly efficient separation of amino acid phenylthiohydantoins by reversed-phase high-performance liquid chromatography

A rapid and efficient separation of amino acid phenylthiohydantoins by high-performance liquid chromatography has been accomplished by step-gradient elution with use of a mobile phase of acetate-buffered aqueous acetonitrile and an octadecylsilyl stationary phase. Typical analyses are completed in less than 12 min. Peak elution positions in this procedure are highly reproducible (with about 0.2% variance) and allow unambiguous identification of all residues. A procedure for the optimal positioning phenylthiohydantoin-Arg and -His is given. Molar extinction coefficients at 254 nm, which are particularly useful with common fixed wavelength detectors, are given for 25 amino acid phenylthiohydantoins.     

O-methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

The mass spectra of aldoses, partially methylated aldoses, deoxyaldoses, and ketoses containing 3–7 carbons, were recorded on the ethers of the trimethylsilyl O-methyl oxime derivatives. Each compound gave a distinctive spectrum indicating the carbon-chain length and the location of substituents. The gas-liquid chromatographic properties of most compounds in this study were also examined.     

Free metal ion depletion by "Good's" buffers. III. N-(2-acetamido)iminodiacetic acid, 2:1 complexes with zinc(II), cobalt(II), nickel(II), and copper(II); amide deprotonation by Zn(II), Co(II), and Cu(II)

Potentiometric, visible, infrared, electron spin, and nuclear magnetic resonance studies of the complexation of N-(2-acetamido)iminodiacetic acid (H2ADA) by Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) are reported. Ca(II) and Mg(II) were found not to form 2:1 ADA2- to M(II) complexes, while Mn(II), Cu(II), Ni(II), Zn(II), and Co(II) did form 2:1 metal chelates at or below physiological pH values. Co(II) and Zn(II), but not Cu(II), were found to induce stepwise deprotonation of the amide groups to form [M(H-1ADA)4-(2)]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed on the basis of various spectral data.