Fluorescamine preparation

Preparation of Reagent and Labelling Procedures. The structure of F-D [2-(2,4-diazobicyclo-2,2,2-octyl)-4-(5-aminofluoresceinyl)-6-morpholinyl 1,3,5-triazine] has been confirmed by its FAB-MS, IR, and H-NMR spectra (9). Briefly, F-D was synthesized by the treatment of fluorescamine isomer I with cyanuric chloride, then reaction with morpholine and DABCO (l,4-diazobicyclo-2,2,2-octane), as illustrated... 

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

Read, M.L., Etrych, T., Ulbrich, K., Seymour, L.W. (1999). Characterisation of the binding interaction between poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery vectors. FEBS Lett., 461(1-2), 96-100. 

The derivatization reaction is performed at ambient temperature by simply mixing the aqueous sample extract witli a phosphate buffer of appropriate pH and then adding the fluorescamine solution in acetonitrile under vigorous stirring. Acetonitrile is the solvent of choice for preparing fluorescamine solutions, because tlie net fluorescence decreases witli a decrease in polarity of the organic solvent in the order acetonitrile, acetone, dioxane, and tetrahydrofuran. 

Glycine was analyzed by the fluorescence of its fluorescamine derivative with excitation at 366 nm and emission at 480 nm (7). A standard working curve prepared simultaneously with the analyte permitted quantitation. 

Fluorescamine stain 0.25 mg/ml fluorescamine (Sigma) in sodium bicarbonate solution (prepare fresh daily)... 

Fluorescamine reacts instantaneously with the primary amino groups of peptides, yielding a fluorescent product with an excitation peak at 390 nm and an emission band at 475 nm. It is insoluble in water and is usually prepared in acetone. Neither the reagent nor the degradation products of the excess reagent in an aqueous medium are fluorescent, which is a great advantage, particularly when postcolumn derivatization is used. 

Moser et al. (1988) described a fluorometric method of detection of lysozyme down to levels of 0.1 /ag. They used highly purified cell walls to prepare a peptidoglycan which is labeled with fluorescamine. After... 

Prepare fluorescamine staining solution by dissolving 5.0 mg fluorescamine in 1.0 ml dimethyl sulfoxide. This solution is very sensitive to light. Therefore, the container used for its storage should be wrapped in aluminum foil. 

Prepare unknown samples, if they exist, in the same manner. 6-54. Repeat step 6-24. Cool the samples back to room temperature. 6-55. Add 0.030 ml of the fluorescamine solution prepared in step 6-50 to each of the cooled samples from step 6-54. Mix the samples as vigorously and quickly as possible after adding fluorescamine so that reaction between the stain and proteins occurs before the reagent decomposes. 6-56. Add 0.005 ml tracking dye (step 6-5) to the solution from step 6-55. 6-57. Repeat step 6-26. 

Sample preparation 1 mL Serum or homogenized tissue + 4 mL MeCN, vortex, centrifuge at 1000 g for 15 min. Remove the supernatant and evaporate it to dr5mess undfer a stream of nitrogen at 40°, reconstitute the residue in 50 xL water, mix vigorously, add 1 mL MeCN, centrifuge at 1000 g for 15 min. Remove the upper layer and evaporate it to dryness, reconstitute the residue in 1 mL 10 ng/mL sulfadiazine in 0.01% trichloroacetic acid, shake, add 100 p,L hexane, shake, centrifuge at 1000 g for 15 min. Remove a 500 p,L aliquot of the clear aqueous layer and add it to 100 p,L 1 mg/mL fluorescamine in MeCN (freshly prepared), shake by hand for 1 min, inject a 50 p,L aliquot. 

Alternatively, chromatography columns can be monitored on line. Fluorescamine and o-phthalaldehyde are ideally suited for monitoring columns in this fashion, because they react rapidly and because the reagents are themselves nonfluorescent. Whereas fluorescamine reacts with both the a-amino graoup of the peptide and the -amino group of lysine residues, o-phthalaldehyde interacts only with those peptides that contain lysine residues (Joys and Kim, 1979). For preparative work, the column effluent is split so that only a portion is utilized for the fluorogenic reaction, and the remainder is collected. This can be accomplished with a discontinuous stream-sampling value, which directs aliquots of column effluent, at predetermined intervals, into the detection stream (Fig. 3) (Bohlen et a/., 1975). In this system, the proportion of column effluent taken for the fluorescence assay can be readily... 

The reaction products of some amines and peptides with fluorescamine have been synthesized as crystalline solids [232], Solutions of these compounds are stable in neutral and mildly alkaline medium in the dark. In acidic solutions the fluoroescence rapidly deteriorates. Fluroescence efficiency is highest at acidic pH because of the highly fluorescent protonated form of the fluorophore. In freshly prepared solutions, the fluorescence intensity decreases in two distinct steps with increasing pH, reflecting the presence of two dissociable acidic functions in the molecule with pK values of 3.8 and 11.6. 

Alkaline hydrolysis was carried out in order to remove PS and PE that can be extracted together with the psychosine. After the alkaline hydrolysis, the extract was washed three times with upper phase containing ammonium hydroxide. If the extract (checked by TLC) had still some contaminants as cerebrosides or sulphatides, a preparative TLC was carried out using chloroform methanol 2 N-NH3 60 35 8 as solvent in order to remove them. The spots were identified by spraying the plate with fluorescamine reagent in acetone (4-phenyl-spiro fu-ran-2 (3H) I -phtalan 3,3-dione) Hoffmann La Roche, Nut ley, U.S.A. 

Detector UV 270 F ex 395 em 495 following post-column reaction. The column effluent mixed with ice-cold reagent pumped at 0.3 mL/min and this mixture flowed through a 2.3 m X 0.5 mm ID coil in a cooled ultrasonic bath to the detector. (Prepare reagent by dissolving 25 mg fluorescamine in 25 mL MeCN and adding 75 mL buffer and 200 -L mercaptoethanol. The buffer was 20 mM sodium dihydrogen phosphate adjusted to pH 3 with 1 M phosphoric acid.)... 

A sensitive fluorometric assay for lysozyme uses as the substrate Bacillus subtilis cell walls that have been labelled with fluorescamine on the free amino-groups of the diaminopimelic acid residues. The method is particularly suitable for measuring the competition between cell-wall preparations for the same enzyme. 

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