Spectroscopy and Fluorimetry
Joseph Chamberlain in The Analysis of Drugs in Biological Fluids, 2018
Dansyl chloride (dimethylaminonaphthalene sulfonyl chloride), is a popular reagent for the formation of a fluorescent conjugate with primary and secondary amines or hydroxyl groups. The reagent was first developed as a fluorescent label for protein studies. The quantum yield of fluorescence is the same for all amino groups (60%) but is only about 10% for phenols. The dansyl chloride reagent was used to determine praziquantel in plasma and urine. In the method described by Pütter381 the drug is extracted from alkaline plasma with a benzene-hexane mixture (1:4). The organic layer is reduced in volume and then successively washed with dilute alkali and dilute acid. The mixture is then hydrolyzed with strong alkali to expose the amine functions which can then react with the dansyl reagent (Figure 4.24). Although in theory two dansyl residues could be coupled to the praziquantel hydrolysis product, it is thought that only one residue is coupled in this case. Other dansyl reagents used in drug analysis include dansyl hydrazine for ketones382 and dansylaziridine for sulfhydryl compounds.383
Methods for Sequence Determination
Roger L. Lundblad in Chemical Reagents for Protein Modification, 2020
The aliquots removed at each cycle are dried in a vacuum desiccator over P2O5. Two successive 2-μl portions of 0.1 M NaHCO3 are added and dried to remove ammonia. Dansyl chloride solution (1 μl of a 1:1 mixture of water and a solution of 2.5 mg dansyl chloride per ml acetone) is added and the sample incubated at 37°C for 1 h. After drying, 5 μl of 6 N HCl is added. The tube is sealed and the peptide is hydrolyzed at 105°C for 16 h. The dansyl amino acids are identified by 2-dimensional thin-layer chromatography on 5 x 5 cm polyamide sheets.10 A fine capillary is used for sample application to keep the spot as small as possible. A mixture of standards is run on the reverse side of the sheet. The solvent for the first dimension is 1.5% (v/v) formic acid. In the second dimension benzene/acetic acid 9:1 (or less toxic toluene/acetic acid 10:1) is run, followed in the same direction by ethyl acetate/methanol/acetic acid 20:1:1. The plate is dried and examined under UV light after each step. Resolution of certain spots may require a fourth solvent, 0.05 M trisodium phosphate/ethanol 3:1, also run in the second direction. As in the subtractive method, glutamine and asparagine are deamidated during hydrolysis. The thiazolinones may be recovered from the organic solvent, converted to the phenylthiohydantoins, and identified by one of the methods described later.
Phosphonic Acids In Nature
Richard L. Hilderbrand in The Role of Phosphonates in Living Systems, 2018
The AEP has been assumed to be covalently linked to the phosphonoproteins because rigorous conditions have failed to separate it from the other constituents. The structure of that linkage has not been determined and is the primary question concerning these unusual biological molecules. 1-Fluoro-2,4-dinitrobenzene treatment failed to produce the 2,4-dinitrophenyl-AEP derivative which would be formed with a primary amino group on the AEP.5,71 Furthermore, Kirkpatrick and Bishop72 could not form a dansyl chloride derivative without acid hydrolysis of the protein. Thus, the amino group of the AEP may be in a peptide linkage or N-acylated. The absence of fatty acid in the phosphonoprotein indicates that the amino group is not present as a long-chain acyl derivative, although an acetyl derivative is possible since acetic acid would not have been detected in the fatty acid assay.72 A precedent exists for this structure with AEP since Hori and Arakawa54 have identified an acylated ceramide-AEP.
Novel plasma metabolite markers of attention-deficit/hyperactivity disorder identified using high-performance chemical isotope labelling-based liquid chromatography-mass spectrometry
Published in The World Journal of Biological Psychiatry, 2021
Liang-Jen Wang, Wen-Jiun Chou, Ching-Shu Tsai, Min-Jing Lee, Sheng-Yu Lee, Chia-Wei Hsu, Pei-Chun Hsueh, Chih-Ching Wu
The detailed procedure for the dansylation reaction is described in previous studies (Lee et al. 2018; Hsu et al. 2019). Briefly, the metabolite extract from each individual specimen was combined with 12.5 μL sodium carbonate/sodium bicarbonate buffer (0.5 M, pH 9.5), 12.5 μL acetonitrile (ACN), and freshly prepared 12C2-dansyl chloride solution (light isotope; 18 mg/mL in ACN). Following incubation in a shaker at 40 °C for 45 min, the mixture was treated with 5 μL of 250 mM sodium hydroxide to quench the labelling reaction, and then 25 μL of 425 mM formic acid was added to adjust the solution to a pH of 3–4. A pooled metabolite extract was used as a universal metabolome standard (UMS), which was reacted in the presence of homemade 13C2-dansyl chloride solution (heavy isotope) (Lee et al. 2018; Hsu et al. 2019).
Defect-induced electronic states amplify the cellular toxicity of ZnO nanoparticles
Published in Nanotoxicology, 2020
Indushekhar Persaud, Achyut J. Raghavendra, Archini Paruthi, Nasser B. Alsaleh, Valerie C. Minarchick, James R. Roede, Ramakrishna Podila, Jared M. Brown
RAECs were exposed to 20 µg/mL of pristine, annealed, oxidized, or reduced ZnO NPs for 6, 12, and 24 h. The cell fraction was collected and prepared for high-pressure liquid chromatography (HPLC) measurements of the thiol/disulfide couples concentration to determine the cellular redox potential. The procedures were based on that of Jones et al. (Jones and Liang 2009). Briefly, the cell pellet was resuspended in 0.5 mL of 5% perchloric acid (PCA)/0.2 M boric acid/10 μM ϒ-Glu-Cys and then sonicated. Samples were centrifuged at 13,000×g for 2 min and 300 µl of the supernatant was placed in a new tube. The tubes with the pellets were aspirated and stored for protein quantification by bicinchoninic acid (BCA) protein assay. Derivation of samples to extract the thiol/disulfide couples was performed on the collected supernatant. For each sample, 60 μL of 9.3 mg/mL iodoacetic acid was added, and the pH was adjusted to 8.8–9.2 with 1 M KOH. The samples were incubated for 20 min at room temperature after which 300 μl of 20 mg/mL of dansyl chloride was added to each sample. The samples were placed in the dark overnight. For each sample, 500 μL of chloroform was added, vortexed, and centrifuged at 13,000×g for 2 min to separate the aqueous and organic layers. HPLC was performed on an amino column with a Supelcosil™ LC-NH2 25 cm × 4.6 mm, 5 μm column (Supleco, Bellefonte, PA) with an Agilent 1200 HPLC equipped with a fluorimeter. The concentration of glutathione, its disulfide form GSSG, cysteine (Cys), and cystine (CySS) was determined from the scans. The Nernst equation was used to calculate the redox potential of the thiol/disulfide ratio:
Associations between Dietary Fiber, the Fecal Microbiota and Estrogen Metabolism in Postmenopausal Women with Breast Cancer
Published in Nutrition and Cancer, 2021
Ayse G. Zengul, Wendy Demark-Wahnefried, Stephen Barnes, Casey D. Morrow, Brenda Bertrand, Taylor F. Berryhill, Andrew D. Frugé
Assays for 17β-estradiol and estrone in serum were performed in the UAB Targeted Metabolomics and Proteomics Laboratory. Estrogen analyses were determined by isotope dilution HPLC-electrospray ionization-multiple reaction ion mass spectrometry adapted from the method of Tai and Welch (18). 17β-Estradiol and estrone standards were prepared in 0.05% BSA. Sera (500 µl) were diluted 1:1 with MilliQ H2O. Samples and standards were spiked with 0.5 ng/ml 13C6-estradiol (CIL, Tewksbury, MA) internal standard. Diluted samples (1 ml) and standards were loaded onto individual 30 mg Polymeric Strata-X Solid Phase Extraction cartridge columns (Phenomenex, Torrance, CA). The cartridges were washed with MilliQ H2O (1 ml) and 40% methanol (1 ml) followed by elution of 17β-estradiol with 1 ml of methanol. Sample eluents were dried under a gentle stream of N2. Sodium bicarbonate (50 µl, 100 mM, pH 10.5) and dansyl-chloride (50 µl, 1 mg/ml) in acetone were added to samples which were incubated at 60 °C for 10 min. Samples were dried once more under a gentle stream of N2 followed by reconstitution in 100 µl of 40% methanol/0.1% formic acid (FA). Chromatography was carried out using an Ace Excel C18-Aromatic 1.7 µm 50 x 3.0 mm IS column at 50 °C using a 20AD Prominence HPLC (Shimadzu, Kyoto, Japan) in tandem with 6500 Qtrap mass spectrometer (SCIEX, Framingham, MA). LC-MS operation and data collection were under the control of Analyst 1.6.2 software (SCIEX). The mobile phases were composed of (A) 0.1% FA and (B) acetonitrile 0.1% FA; the flow rate was 300 µl/min. Gradient starting conditions were 50% B which was held for 1 min, a linear increase of B to 100% B at 4 min, held at 100% B until 4.75 min, and returned to 50% B at 5 mins, to equilibrate to starting conditions until 7 min. LC flow was diverted to waste for the first 1.8 min to prevent salt contaminating the MS front end. The MS was operated in positive electrospray ionization mode with the following parameters: curtain gas 30, collision gas medium, temperature 500, ion spray voltage 5000, collision energy 25, GS1 60 and GS2 60. Mass transitions for multiple-reaction-monitoring mode were m/z 506/171 for dansyl-17β-estradiol, 504/171 for dansyl-estrone and m/z 512/171 for 13C6-dansyl-17β-estradiol. The standard curve ranged from 5 – 5000 pg/ml over seven points. All data were processed, and concentration factors were corrected using Multiquant 3.0.1 (SCIEX).
Related Knowledge Centers
- Amine
- Dimethyl Sulfoxide
- Fluorescence
- Forster Resonance Energy Transfer
- Protein Sequencing
- Sulfonamide
- Tryptophan
- Amino Acid
- Dansyl Amide
- Förster Resonance Energy Transfer
- Α-Cyclodextrin