Polymeric 6-aminoquinoline, an activated carbamate reagent for derivatization of amines and amino acids by high-performance liquid chromatography

A new polymeric reagent containing the 6-aminoquinoline (6-AQ) tag was developed and applied for the off-line derivatization of amines and amino acids in high-performance liquid chromatography (HPLC). The synthesis and characterization of this polymeric reagent are described. An authentic external standard of a typical amine was synthesized and characterized for the determination of the derivatization efficiency. All amines had a derivatization efficiency higher than 50%; the derivatization of amino acids was performed under optimized phase-transfer catalysis reaction conditions. Derivatized amines and amino acids were separated under conventional reversed-phase conditions and determined by UV and FL detectors. To investigate the practical applications, this polymeric reagent was also used to derivatize protein hydrolysates.     

Stable isotope ratio analysis of amino acids: the use of N(O,S)-ethoxycarbonyl trifluoroethyl ester derivatives and gas chromatography/mass spectrometry

N(O,S)-Ethoxycarbonyl trifluoroethyl amino acid esters are formed by the reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. The use of these derivatives for a rapid and sensitive determination of specific enrichment of stable isotopically labeled tracer amino acids in blood plasma and protein hydrolysates, by using gas chromatography/electron impact mass spectrometry, is discussed.     

Amino acid concentrations in portal venous plasma during absorption from the small intestine of the guinea pig of an amino acid mixture simulating casein and a partial enzymic hydrolysate of casein

1. The characteristics of absorption of individual amino acids from amino acid mixtures simulating casein and from enzymic hydrolysates of casein containing oligopeptides as well as free amino acids are known to be different. The differences, which are attributable to mucosal uptake of small peptides, involve more rapid absorption from the enzymic hydrolysates of certain amino acids which are relatively slowly absorbed from the amino acid mixtures. This could lead to more effective utilization of amino acids from the enzymic hydrolysates than from the amino acid mixtures. 2. To obtain further information bearing on this hypothesis, we have used a recently developed technique for portal cannulation in the guinea pig to make a preliminary investigation of amino acid concentrations in the portal venous plasma at intervals after the infusion into the duodenum of equivalent amounts of (a) an amino acid mixture simulating casein and (b) a partial enzymic (papain followed by kidney peptidases) hydrolysate of casein, the two preparations being infused in separate experiments. 3. For some amino acids, such as leucine, isoleucine, valine, phenylalanine and lysine, the curves after the enzymic hydrolysate were fairly similar to the corresponding curves after the amino acid mixture, though usually slightly lower. With other amino acids, the curves after the enzymic hydrolysate were very much lower than the corresponding curves after the amino acid mixture. With serine, glutamine, proline and glycine this discrepancy was particularly great. 4. The results cannot yet be fully explained, but their main features are explicable by the hypothesis that the lower amino acid concentrations in portal plasma after the enzymic hydrolysate are the result of entry of amino acids into the portal blood in peptide form, in which they would not be detectable by the analytical technique employed, and possibly also of more rapid clearance of amino acids from the blood during absorption of this preparation.     

Novel histamine measurement by HPLC analysis used to assay histidine decarboxylase inhibitory activity of shoyuflavones from soy sauce

An easy and highly sensitive method for measuring histamine by HPLC analysis coupled with precolumn derivatization was established. The amino group of histamine was completely colorimetrically labelled with 4-N,N-dimethylamino-azobenzene-4'-isothiocyanate (DABITC) in the presence of sodium bicarbonate at 90 degrees C for 5 min. The derivative was sensitively and easily analyzed by HPLC on a Cosmosil 5SL column using CHCl3/N,N-dimethylformamide/H2O (210:90:4) containing 0.4% acetic acid. Using the established method, histidine decarboxylase (HDC) inhibitory activities of three tartaric acid isoflavone derivatives, named shoyuflavones, isolated from soy sauce were examined in vitro by measuring the histamine produced by HDC. They showed intense inhibition of the activities of HDC from both mouse mastocytoma P-815 cells and Clostridium perfringens.     

New amino-nitroxide spin labels

Stable free mono- and diradicals containing reactive primary or secondary amino groups in the side-chain have been synthesized by transesterification of amino-substituted esters with paramagnetic alcohols or from spin-labeled acid derivatives and amines. In the second approach the new radical 18 (1-oxyl-3-(2-bromoethoxycarbonyl)-2,2,5,5-tetramethylpyrroline) is proposed as an efficient alkylating species. The nitroxides described are pH-sensitive spin probes and spin labels potentially useful for a diversity of ESR applications in chemistry and biology. New spin-labeled tyramine 16 (N-(1-oxyl-3-carbonyl-2,2,5,5-tetramethyl-pyrroline)tyramine) was successfully employed in a novel assay of protein oxidative damage.     

Selective determination of secondary amines as their N-diethylthiophosphoryl derivatives by gas chromatography with flame photometric detection

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o-phthaldialdehyde, secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB-1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05-0.2 pmol per injection. N-Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1-20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean-up. Overall recoveries of secondary amines added to urine samples were 91-105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.     

Synthesis and antibacterial action of new ureido- and dicarboxylic acid diamido- derivatives of acylpenicillins with and without catechol substituents

New ureido, oxamoyl, fumaramoyl and terephthalamoyl derivatives of ampicillin or amoxicillin were synthesized by reaction of acylpenicillines with o-dihydroxy- or o-diacyloxy substituents containing aromatic amines bound over CO- or dicarboxylic groups. Corresponding compounds derived from 3,4-diacetoxyaniline showed significant increase of activity against pseudomonas and salmonella in contrast to derivatives without catechol substituents. No increase of activity was observed by corresponding derivatives of bi- and tricyclic amines. Derivatives with oxamoyl, fumaramoyl or terephthalamoyl groups were found to be more active than the corresponding ureido derivatives. Studies with mutants possessing higher membrane permeability have shown that the high activities of catechol containing derivatives are connected with the improved penetration through the outer membrane. Some new penicillin derivatives are more stable against beta-lactamases compared with azlocillin.     

Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100x1 mm I.D. C8, 5 microm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 microl/min. For monoamines and metabolites we used a 150X1 mm I.D. C18 5 microm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 microM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-microm tissue slices. Tissue samples were homogenized in 50 or 100 microl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and gamma-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Electron capture gas chromatographic analysis of the amine metabolites of pesticides: derivatization of anilines

A number of amines have been shown to result from metabolism of various pesticides. From an epidemiological standpoint, it may be possible to monitor human exposure to these pesticides through the excretion of their corresponding amines in urine. An investigation has been initiated to develop and apply methods of analysis of amines in human urine. The results of a survey of derivatization techniques involving several substituted anilines are presented. These include conditions for derivatization, utilizing a number of halo- and nitro- substituted reagents; electron capture and gas chromatographic properties of the derivatives; and stability of the derivatives to extraction and column chromatography for purposes of separation and cleanup. The recoveries of anilines from spiked water and urine samples at the 1.0 ppm and 0.1 ppm levels were between 85 and 90%. The advantages and disadvantages of the various derivatives and techniques are discussed and a rationale is presented for the preliminary selection of a particular derivative for application of the analysis of aniline metabolites in urine.     

A single-column amino acid analysis method which resolves hexosamines and several cysteine derivatives

A single-column amino acid analysis method is presented for use in structural studies of glycoproteins. The system gives excellent resolution of glucosamine, galactosamine, cysteic acid, CM-cysteine, AE-cysteine, the internal standard norleucine, and all amino acids normally present in protein hydrolysates.     

Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis

A sensitive method for quantitatively determining submicromolar levels of neurotransmitter amino acids (e.g. Asp, Glu and gamma-aminobutyric acid) in microdialysates from brain and cerebrospinal fluids is reported. 6-Aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) was employed as the derivatization reagent, followed by HPLC separation and fluorescence detection of the derivatives. The derivatization was conducted simply by mixing the AQC directly with the microdialysis samples. The reaction was complete within seconds after mixing at room temperature. Separation development optimizing the gradient profile, eluent pH and column temperature resulted in an excellent separation of the required amino acids in less than 30 min. Other resolved amino acids in the same profile include Gly, taurine, and Pro. Recoveries for the amino acids of interest spiked into high salt containing perfusion buffers were greater than 97%. The sensitivity of the method was increased by employing a 16-microliter flow cell in the detector and analyzing 20-microliter aliquots of the derivatization mixtures. With the optimized conditions, the detection limits were 3-7 nM (fmol/microliter). Typical reproducibility (%R.S.D.) for quantitation of these amino acids at submicromolar levels was approximately 2%. Excellent linearity (r2 > 0.999) was achieved over the range 0.2-20 microM. The low detection limits permitted the analysis of a number of different microdialysate samples including those from cerebrospinal fluid, as well as substantia nigra and hypothalamus from brain samples, even at basal levels where gamma-aminobutyric acid concentration may be < 50 nM. The excellent sensitivity made it easy to distinguish basal from stimulated levels of neurotransmitter amino acids, even from sample sizes as small as 10 microliters.     

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6-150 pg injected. The calibration curves for each amino acid in the range of 0.02-2 micrograms were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83-112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3-8.9% (n = 3) and 1.9-15.8% (n = 3), respectively.     

2-Methyl-3-oxo-4-phenyl-2,3-dihydrofuran-2-yl acetate: a fluorogenic reagent for detection and analysis of primary amines

A new fluorogenic reagent, 2-methyl-3-oxo-4-phenyl-2,3-dihydrofuran-2-yl acetate, has been developed for the analysis of primary amines and aminated carbohydrates by means of HPLC, CE, and MALDI/MS. Peptides at 1 pmol (2 x 10(-7) M) levels were successfully labeled and analyzed through CE. The fluorescent derivatives have good stability in both acidic and basic solutions, making their further manipulation and structural analysis possible. The derivatives can be analyzed in reversed-phase HPLC due to the hydrophobic nature of this fluorescent tag. Characteristic elution intervals between the diastereomeric peaks of the chiral peptide derivatives may be used in structural verification. The labeled peptides and neutral oligosaccharides are also readily detectable through MALDI/MS in its positive mode.     

Phenol-degrading denitrifying bacteria in wastewater sediments

The simultaneous determination of amino and organic acids in plant tissue extracts using capillary gas chromatography is described. Plant leaves were extracted in 5% (w/v) perchloric acid and neutralized extracts were purified using C18 cartridges. The amino and organic acids in purified extracts were then converted to tert-butyldimethylsilyl (TBDMS) derivatives prior to separation and detection by capillary gas chromatography (GC) with flame ionization detection. Conditions required for optimal derivatization were investigated. Amino and organic acids were readily converted to their TBDMS derivatives using N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide in dimethylformamide solvent 1:6 (v/v) with an average recovery of 90% and a reproducibility of about 5%. The characteristic [M-57] and [M-159] fragment ion of the TBDMS derivatives was confirmed using GC-MS. The proposed method was demonstrated by the determination of amino and organic acids in extracts of Acacia and Eucalyptus leaves, where detection limits were 1-20 ng.     

2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins

Cyanomethyl 1-thioglycosides ofD-galactose, D-glucose, 2-acetamido-2-deoxy-D-glucose, and D-mannose were prepared from the respective pseudothiourea derivatives and chloroacetonitrile. The nitrile group in these cyanomethyl thioglycosides can be converted to a methyl imidate group by treatment with sodium methoxide or HC1 in dry methanol to yield 2-imino-2-methoxyethyl 1-thioglycosides (IME-thioglycosides). The factors influencing the yield of IME-thioglycosides were investigated. The most convenient method of preparing IME-thioglycosides was treating 0.1 M cyanomethyl thioglycoside peracetate in dry methanol with 0.01 M sodium methoxide at room temperature for 24-48 h (50-60% yield). These IME-thioglycosides reacted readily with simple amines, amino acids, and proteins in mildly alkaline buffer solutions. Alpha-amylase and lysozyme modified with these reagents under appropriate conditions retained full activities. Thus the IME-thioglycosides constitute a new group of reagents for attaching sugars to proteins.     

Tryptophan in bovine rhodopsin: its content, spectral properties and environment

The tryptophan content of purified bovine rhodopsin was obtained by two independent methods: direct analysis of hydrolysates prepared by digestion of opsin with methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole and a computer-assisted analysis of the near-UV spectrum of rhodopsin. Both methods gave a value of eight tryptophan residues per rhodopsin. Based on the near-UV spectral analysis, the light-induced difference spectrum of rhodopsin, and the susceptibility of residues to oxidation by N-bromosuccinimide, we concluded that approximately half of the tyrosine and tryptophan residues are shielded to some extent from the aqueous solvent, that two of the tryptophan residues are in very apolar environments, and that following light excitation at least one of these tryptophan residues and several tyrosines are exposed to an aqueous environment. Analysis of rhodopsin absorption in the far-UV indicated that below 240 nm, approximately half of the absorption is due to aromatic residues and that the other half is largely due to the peptide bond. The effect of illumination on secondary structure is to induce a loss in helical structure, calculated to involve 35% of the amino acid residues in purified rhodopsin. If light-induced changes in secondary structure are specifically excluded, most of these results can be extended to bovine rod outer segment membranes.     

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.     

Amino acid analysis by gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters. Complete resolution using a support-coated open-tubular capillary column

Amino acids have been separated by gas-liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenaschuk have been introduced. Acylation by heating at 150 degrees was shown to be destructive; 110 degrees has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of beta-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxy-peptidase digestion.     

Determination of free and total proline and hydroxyproline in plasma and tissue samples by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of free and total proline (Pro) and 4-hydroxyproline (Hyp) by gas chromatography (GC) was developed. For free Pro and Hyp analysis, plasma and tissue homogenate were extracted with methanol. For total Pro and Hyp analysis, these samples were hydrolysed in 6 M HCl. After removal of primary amino compounds by the reaction with o-phthaldialdehyde, Pro and Hyp in methanol extract and acid hydrolysate were converted into their N-dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB-5 capillary column. This method was successfully applied to small samples without prior clean-up, and Pro and Hyp in these samples could be analysed without any influence from coexisting substances. Overall recoveries of Pro and Hyp added to plasma and tissue samples were 92-106%. The analytical results of free and total Pro and Hyp in human plasma and mouse tissue samples are presented.     

Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 mug of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio. The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radio-activity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel.