Organic fluorescence reagents in the study of enzymes and proteins.
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Affinity Labels↗
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Results for “Dansyl Compounds”
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[Dansyl chloride as a reagent for sympathomimetic amines, I (author's transl)].
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Dansyl Compounds↗
[Dansyl chloride as a reagent for sympathomimetic amines, II: Fluorimetric determination of ephedrine hydrochloride as its dansyl derivative in tablets (author's transl)].
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Dansyl Compounds↗
Role of the tissue factor pathway in synovial inflammation.
OBJECTIVE: Clinical and experimental evidence suggests that extravascular fibrin deposition in arthritic joints is prominent and deleterious. The aim of this study was to investigate the contributions of tissue factor (TF) and its inhibitor, TF pathway inhibitor (TFPI), in arthritis. METHODS: Synovial tissue specimens obtained from 10 patients with rheumatoid arthritis (RA) and 12 patients with osteoarthritis (OA) were scored histologically for inflammation and fibrin content. TF and TFPI levels were assayed at antigenic and functional levels. TF messenger RNA (mRNA) levels were determined using RNase protection assays. The effect of TF inhibition in murine antigen-induced arthritis (AIA) was assessed by administering systemically active site-blocked activated factor VIIa (FVIIai). RESULTS: Functional TF activity was significantly increased in synovial membranes from RA patients compared with those from OA patients. In contrast, no difference in TF mRNA and TF antigenic levels was observed between these 2 groups. This discrepancy can be accounted for by TFPI, because we observed a negative correlation between TF activity and TFPI activity. There was a significant difference between the RA and OA groups in terms of synovial inflammation, with more inflammation observed in the RA group. Most importantly, TF activity was associated with fibrin (P = 0.024) and with histologic inflammation (P = 0.03) scores. In AIA, inhibition of TF-induced coagulation by FVIIai led, on day 9 of arthritis, to decreased synovial thickness and decreased articular cartilage damage, although only the latter difference between controls and treated mice reached significance (P < 0.04). Finally, in FVIIai-treated mice, there was a strong negative association between the prothrombin time and intraarticular fibrin deposition. CONCLUSION: Our results show that TF expression in arthritic synovial tissue favors extravascular coagulation and may play a role in inflammation in RA. In this context, TF inhibitors may be of therapeutic value.
Aged↗
Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids.
Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74 microliter flow cell (originally 10 microliters in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100 microns i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N = 2) of 200 fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).
Amino Acids↗
Fluorometric analysis of urinary chondroitin sulfate isomers by HPLC using dansylhydrazine as a prelabeling reagent.
Chondroitin sulfate isomers (ChS isomers) were digested with chondroitinase ABC, and the unsaturated disaccharides produced were converted into fluorescent dansylhydrazine derivatives and analyzed by HPLC. The proposed method was applied to the determination of relative amounts of ChS isomers in normal human urine and analytical results showed that chondroitin-6-sulfate and chondroitin-4-sulfate were excreted in large amounts. Moreover, this method was used for the identification of urinary ChS isomers which were extracted from a cellulose acetate strip after carrying out electrophoresis.
Chondroitin↗
Thin layer chromatography of dansyl and dinitrophenyl derivatives of amino acids. A review.
The dinitrophenyl (DNP) derivatives of amino acids have found continual application in protein sequencing since Sanger used them for the first time for the sequencing of insulin. Dansyl derivatives of amino acids have been widely used in protein sequencing because of their fluorescent nature. The success of protein sequencing largely depends upon correct identification of such derivatives. The choice for the method of identification is related to cost, the availability of instrumentation and to the sensitivity needed for the analysis. Thin layer chromatography (TLC) is simple and has several advantages over other chromatographic methods. Therefore the literature after 1972 is reviewed for TLC analysis of dansyl- and DNP-amino acids, the two important amino acid derivatives required for identifying protein sequences. Additionally, the literature on the TLC resolution of enantiomeric mixtures of dansyl amino acids is reviewed. Application of various adsorbents, composition of solvent systems and other experimental conditions together with successful resolution data have been discussed. TLC provides a direct and inexpensive method for the resolution of enantiomers, and is fast becoming a sensitive instrumentalized quantitative analytical technique.
Amino Acids↗
A sensitive determination method for mexiletine derivatized with dansyl chloride in rat plasma utilizing a HPLC peroxyoxalate chemiluminescence detection system.
A sensitive determination method for a non-fluorescent anti-arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO-CL) detection system. After an internal standard (4-methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 microL rat plasma, the solution was poured onto an Extrelut 1 column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH 8.5) and mixed with dansyl chloride (75 nmol) in acetronitrile. After standing of 90 min at room temperature, 0.5 N HCl was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO-CL detection system using bis(4-nitro-2(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20-100 ng/mL plasma (20.6-103 fmol/injection). The detection limit (S/N = 2) was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug [25 mg (115.9 mumol)/kg] to rats.
Animals↗
Derivatization with fluorogenic benzofurazan reagents of amino acid enantiomers and their separation on a Pirkle type column.
L- and D-Amino acids (Leu or Phe) were derivatized with fluorogenic reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and 5-(N,N-dimethylamino)naphthalene-1-sulfonylchloride (DNS-CI), and separated on a Pirkle type column, Sumichiral OA 2500 (S) ((S)-1-naphthylglycyl-3,5-dinitrophenylamide silica gel) with a mobile phase of 20 mM ammonium acetate in methanol. The fluorometric detection of the derivatives was made at 530 nm, 590 nm, 590 nm and 530 nm with excitation at 470 nm, 450 nm, 450 nm and 350 nm, respectively. The former three derivatives of the enantiomers were separated well from each other; The alphas for each NBD-, DBD- and ABD-derivative of L- and D-Leu were 1.10, 1.11 and 1.10, respectively. However, the DNS derivatives of L- and D-Leu were not separated (separation factor, alpha = 1.0). All NBD-, DBD- and ABD-derivatives of L- and D-Phe were also well separated (alphas were 1.18, 1.17 and 1.16, respectively), while DNS-L- and -D-Phe were barely separated (alpha = 1.04). These data suggest that the 2,1,3-benzoxadiazole (benzofurazan) moiety is very effective and preferable to the dimethylaminonaphthalene sulfonyl (DNS) structure for the separation of enantiomers of amino acids derivatized with benzofurazan reagents.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high-performance liquid chromatography.
We have optimized a method for the determination of intracellular glutathione by high-performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status.
Animals↗
Development and validation of a high-sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with chemical derivatization for the determination of ethinyl estradiol in human plasma.
An ultra-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of oral contraceptive ethinyl estradiol (EE) was developed and validated over the curve range of 2.5-500 pg/mL using 1 mL of human plasma sample. Ethinyl estradiol and the internal standard, ethinyl estradiol tetra-deuterated (EE-d4), were extracted from the plasma matrix with methyl t-butyl ether, derivatized with dansyl chloride and then back-extracted into hexane. The hexane phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatographic separation was achieved on a Luna C(18) column (50 x 2 mm, 5 micro m) with an isocratic mobile phase of 20:80 (v/v) water:acetonitrile with 1% formic acid. The offline derivatization procedure introduced the easily ionizable tertiary amine function group to EE. This greatly improved analyte sensitivity in electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 2.5 pg/mL. This high sensitivity method can be used for therapeutic drug monitoring or supporting bio-equivalence and drug-drug interaction studies in human subjects.
Chromatography, Liquid↗
Determination of eugenol in rat plasma by liquid chromatography-quadrupole ion trap mass spectrometry using a simple off-line dansyl chloride derivatization reaction to enhance signal intensity.
A rapid, selective and sensitive method was developed for the determination of eugenol concentration using an off-line dansyl chloride derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with dansyl chloride and analysis by full scan liquid chromatography electrospray quadrupole ion trap mass spectrometry (LC-ESI-QIT). The separation was achieved using a 100 x 2 mm C(8) analytical column combined with an isocratic mobile phase composed of 75:25 acetonitrile: 0.1% formic acid in water set at a flow rate of 0.25 mL/min. Signal intensity of the eugenol-dansyl chloride derivative was increased up to 100-fold as compared with the underivatized eugenol in positive electrospray mode. An analytical range of 100-20,000 ng/mL was used in the calibration curve of plasma and blood samples. The LOD observed was 0.5 pg injected on column. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. In conclusion, a rapid and sensitive LC-ESI/MS/MS method using a derivatization agent was developed to enhance signal intensity of eugenol.
Animals↗
Detection and quantitative analysis of amphetamine.
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Amphetamine↗
A method of analysis of dansylamides in mixtures by mass spectrometry.
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Amides↗
Some new fluorescent derivatives for the mass spectrometric quantitation of biogenic amines.
The 5-dimethyl-, diethyl-, dipropyl-, dibutyl-, and dipentyl-aminoaphghalene-1-sulfonyl (dansyl, ethanyl, propansyl, bansyl and pentansyl respectively) derivatives of tyramine and other biogenic amines were prepared and their mass spectra recorded. The relative intensity of the largest unique ion increased with increasing length of the alkyl group. Several O-alkyl-N-propansyl- and N,O-dialkyl-N-propansyl-, bansyl- and pentansyl-tyramines were also synthesized and their mass spectra recorded. Dimethylbansyl- and dimethylpentansyl-tyramines exhibited the largest unique ions in their mass spectra and the greatest sensitivity in quantitation by the integrated ion current method. Procedures for preparing these derivatives in amounts ranging from nanograms to milligrams are presented and their thin-layer chromatographic behavior in three solvent systems is described.
Biogenic Amines↗
Profiling and quantification of biogenic amines in wine by constant B2E linked scan mass spectrometry.
The constant B2E linked scan is shown to be an excellent tool for the profiling of biogenic amines in wine samples. The fragment ion at m/z 169 of the dansylated amines is used for the precursor ion search in combination with direct sample evaporation. Besides the profile a semi-quantification may be achieved when using suitable amines as internal standards with standard deviations of 12-17%. More accurate results with standard deviations of 3-5% are obtained when using deuterated analogues as internal standards. Both methods are applied to the analysis of biogenic amines in synthetic mixtures and actual wine samples.
Biogenic Amines↗
Factors affecting the fragmentation of peptides in fast atom bombardment mass spectrometry.
Positive ion fast atom bombardment mass spectra of a series of biologically active peptides were obtained in order to ascertain conditions that contribute to an increase in fragment ion abundance of such peptides. The effect of derivatization, hydrophobic character and the presence of acid in the matrix on fragmentation of peptides were all investigated. It is shown that a substantial increase in fragment signal ion abundance is observed when mineral acid is present in the matrix, and is in accord with the gas-phase collision model proposed by Kebarle.
Acetylation↗
Metabolism of polyamines and basic amino acids in Erwinia amylovora: application of liquid chromatography/electrospray mass spectrometry to proferrioxamine precursor feeding and inhibition studies.
Erwinia amylovora, the etiological agent of fire blight, produces a family of proferrioxamine siderophores, which may be essential for the pathogen to establish itself in its hosts. If so, then control of fire blight may perhaps be possible via interference with proferrioxamine biosynthesis. Proof of this hypothesis requires prior knowledge of the corresponding biosynthetic pathways in E. amylovora. As a first step towards understanding proferrioxamine biosynthesis, it was of interest to investigate the ability of the fire blight bacterium to utilize various potential biosynthetic pathways for diamines. Feeding of lysine, ornithine and diaminobutyric acid gave rise to highly elevated levels of cadaverine, putrescine and diaminopropane, respectively, indicating that the corresponding decarboxylase activities are all present in E. amylovora. The conclusion for lysine decarboxylase was confirmed with (15N2)lysine, which was converted to (15N2)cadaverine. Arginine did not increase putrescine levels substantially, but (13C6)arginine nevertheless gave rise to (13C4)putrescine while suppressing excretion of non-labeled putrescine. A serendipitous result of this study was the finding that the growth of E. amylovora can be inhibited with 5-hydroxylysine and 1,4-diamino-2-butanone. The mechanism of inhibition appears complex and is not yet understood. For 5-hydroxylysine, preliminary investigations point to a competitive inhibition of lysine decarboxylase. However, the growth inhibition cannot be reversed by providing cadaverine, the decarboxylation product of lysine.
Amino Acids↗