PubMed · 9116822
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J M Walker. 1997. The Dansyl-Edman method for manual peptide sequencing.. https://doi.org/10.1385/0-89603-353-8%3A183
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A new derivatization procedure has been developed where solid-phase catalysis is utilized to facilitate the formation of hydrazones in precolumn labeling of keto-containing compounds. This procedure has been implemented on a solid-phase enrichment and enhanced derivatization (SPEED) device, prepared from porous polyethylene that has been coated with Nafion and dansylhydrazine. The SPEED devices have been optimized using experimental design and characterized for dansylation of C-21 ketosteroids by multivariate data analysis, using progesterone as the model compound. The reaction temperature and the molar ratio between the steroid and the derivatization reagent were found to be the factors most strongly affecting the reaction. Faster reaction kinetics were achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the four derivative peaks eluting when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti mono- and bis(hydrazones) formed in the reaction. Using optimal reaction conditions, the derivatives mainly constitute the syn and anti conformers of bis-derivatives. In contrast to solution-based acid catalysis, the SPEED device was remarkably insensitive to water in the reaction mixture. A sample volume of 400 microL was found to be the maximum, enabling sample enrichment prior derivatization. Using optimal experimental conditions, picomole amounts of ketosteroids could be derivatized in 10 min at room temperature. Analysis of spiked serum samples containing 0.4-2.0 nmol of progesterone showed overall recoveries of 52-63%. The corresponding 3delta detection limit was 1.3 pmol (n = 4, 100 microL injected), as estimated from calibration curve data.
A novel fluorophore viz. 4-dansylamido-1,8-naphthalimido-N-pentanol has been designed, prepared and characterised. The comparative fluorescence has been studied in different solvents, solvent gradient, aqueous solutions of inorganic ions and buffers. This can be used for covalent tagging of oligonucleotides having potential application in Molecular Biology.
The effect of sulfatide on membrane hydration of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations >/=30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.