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PubMed1978

Participation of X47-fluorescamine modified E. coli tRNAs in in vitro protein biosynthesis.

The reaction of fluorescamine with primary amino groups of tRNAs was investigated. The reagent was attached under mild conditions to the 3'-end of tRNAPhe-C-C-A(3'NH) from yeast and to the minor nucleoside x in E. coli tRNAArg, tRNALys, tRNAMet, tRNAIle and tRNAPhe. The primary aliphatic amino groups of these tRNAs react specifically so that the fluorescamine dye is not attached to the amino groups of the nucleobases. E. coli tRNA species modified on the minor nucleoside X47 can all be aminoacylated. An involvement of the minor modified nucleoside X47 in the tRNA: synthetase interaction is detected. Native tRNALys-C-C-A from E. coli can be phenylalanylated by phenylalanyl-tRNA synthetase from yeast, whereas this is not the case for fluorescamine treated tRNALys-C-C-A(XF47). Pre-tRNAPhe-C-C-A(XF47) forms a ternary complex with the elongation factor Tu:GTP from E. coli, binds enzymatically to the ribosomal A-site and is active in poly U dependent poly Phe synthesis. Fluorescamine-labelled E. coli tRNAs provide new substrates for the study of protein biosynthesis by spectroscopic methods.

Bacterial Proteins↗
PubMed1997

Variations in pigment and carbohydrate content of gallbladder bile affect accurate quantitation of total protein when using the fluorescamine method.

BACKGROUND: Despite solute dilution and reduced total lipid concentrations, an unexplained increase in protein concentration has been reported to occur in the gallbladder bile of cholesterol gallstone patients. METHODS: Solutes in gallbladder bile from gallstone-free controls and from four study groups were measured using standard methods. Total proteins were measured using amino acid analysis and a conventional fluorescamine method. RESULTS: Bile salts and pigment content were greater in gallstone-free controls than in all other study groups, including morbidly obese gallstone-free subjects. Total biliary protein concentration, as determined by amino acid analysis in the gallstone-free control group was higher than in non-obese gallstone patients with multiple stones and in morbidly obese gallstone-free subjects. Total biliary proteins as measured with fluorescamine, however, did not show intergroup differences. A major problem of the conventional fluorescamine assay is shown to be an artefact arising from the high pigment content of the more concentrated samples. CONCLUSIONS: Very dilute gallbladder bile samples are often found in the presence of gallstone disease. This also occurs in morbidly obese subjects, even in the absence of gallstones. Although the contribution of protein secretion/absorption by the gallbladder can also be relevant, especially in the presence of morbid obesity, the protein concentration in gallbladder bile, when accurately measured, generally parallels the concentrations of non-absorbed biliary solutes, reflecting the efficiency of fluid absorption. Measurement of biliary proteins by the conventional fluorescamine method is unreliable in clinical studies in which intergroup differences in pigment content are commonly present.

Adult↗
PubMedDate not supplied

A comparison of fluorescence versus chemiluminescence detection for analysis of the fluorescamine derivative of histamine by HPLC.

Fluorescence and chemiluminescence detection were compared for HPLC analysis of the fluorescamine derivative of histamine. The kinetic behaviour of the chemiluminescent response for the derivative was characterized in a static system. An HPLC method was optimized for the derivative using fluorescence detection. Fluorescence detection was linear over the range of 166-1666 pg on column for the fluorescamine-histamine derivative with a limit of detection of 13 pg on column. Using a detector designed for optimal use with chemiluminescence, the chemiluminescence response of the fluorescamine derivative was linear over a range of 1.66-16.6 ng on column with a limit of detection of 1.0 ng on column. These results exemplify a case in which superior detectibility is provided by fluorescence over chemiluminescence, and contradicts many reports comparing fluorescence to chemiluminescence. The authors conclude that chemiluminescence should be considered when indicated by conditions established for separation that are favourable for the observation of chemiluminescence. These conditions include sufficiently low excitation energies corresponding to an excitation maximum greater than 400 nm, favourable dipole character of analytes, mobile phases of high organic content, and an appropriate pH of the mobile phase.

Chromatography, High Pressure Liquid↗
PubMed1976

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: a comparative study on the effects of coagulant and non-coagulant fixatives.

The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulant fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.

Amines↗
PubMed2003

Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration.

Chiral separations of fluorescamine-labeled amino acids are characterized and optimized on a microfabricated capillary electrophoresis (CE) device. A standard mixture of acidic and neutral amino acids is labeled with fluorescamine in less than 5 min and the hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentration, temperature, and pH are optimized (15 mM HPbetaCD, 6 degrees C, pH < 9) to achieve high-quality and low background chiral separations in less than 200 s. All four stereoisomers formed in the labeling reaction of the chiral dye with the chiral amino acids are typically resolved. At pH > 9, isomerization of the dye chiral center is observed that occurs on the time scale of the chip separation. Typical limits of detection are approximately 50 nM. These results demonstrate the feasibility of combining fluorescamine labeling of amino acids with microfabricated CE devices to develop low-volume, high-sensitivity apparatus and methods for extraterrestrial exploration.

Electrophoresis, Capillary↗
PubMed1991

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine.

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.

Angiotensins↗
PubMed1998

Postcolumn derivatization of peptides with fluorescamine in capillary electrophoresis.

Fluorescamine is used as a postcolumn derivatization reagent for fluorescence detection detection of peptides after separation by capillary electrophoresis. The problems resulting from the use of an organic solvent have been solved by introducing LiC1O4 and 5% water into the postcolumn derivatization reagent. The reaction rate and detection sensitivity of amino acids and small peptides observed with fluorescamine and OPA were compared. Fluorescamine gives much higher sensitivity than o-phthaldialdehyde (OPA) for small peptides, with detection limits for the selected peptides and amino acids below 0.1 mumol 1-1. Under optimized experimental conditions, the method has a good reproducibility and separation efficiency for peptides. The method was applied for the analysis of the protein tryptic digests. Only submicromolar concentrations of proteins were required.

Amino Acids↗
PubMed1982

Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs. Application to tocainide.

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride-sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol-phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 microgram/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 microgram/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.

Anti-Arrhythmia Agents↗
PubMed1978

Use of fluorescamine-labeled casein as a substrate for assay of proteinases.

Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.

Caseins↗
PubMed1982

A simple and sensitive proteinase assay using Sepharose 4B-coupled fluorescamine-labeled casein as a substrate.

A simple and sensitive method for proteinase assay was developed, which uses fluorescamine-labeled casein-Sepharose 4B as a substrate. Casein-Sepharose 4B was prepared most effectively by coupling casein to cyanogen bromide-activated Sepharose 4B at pH 10.0. Fluorescamine-labeled casein-Sepharose 4B was then prepared by mixing fluorescamine and casein-Sepharose 4B suspension at pH 8.0 and used for the assay as a substrate after removal of the excess reagent and/or its hydrolysis products. The assay can be done by simple measuring the fluorescence (excitation at 390 nm and emission at 475 nm) of the filtrate of the assay mixture after incubation of the substrate with enzyme solution. This method is suited for the assay of proteinases active at neutral to slightly alkaline pH values, and the activity of 3 ng of trypsin or 10 ng of alpha-chymotrypsin can be determined with reasonable accuracy. This method is therefore almost as sensitive as those using radioisotope-labeled proteins as substrates.

Animals↗
PubMed1983

Application of the fluorescamine reaction with 6-aminopenicillanic acid to estimation and detection of penicillin acylase activity.

6-Aminopenicillanic acid may be quantitatively estimated by its reaction with fluorescamine in the concentration range of 1 to 10 micrograms/ml. The difference in reactivity between 6-aminopenicillanic acid and benzylpenicillin, which does not react with fluorescamine, can be used to determine penicillin acylase activity and obtain data on enzyme parameters and inhibitors. Unlike amino acids and peptides, 6-aminopenicillanic acid reacts strongly with fluorescamine at pH 4, an observation which can be used to determine the presence of penicillin acylase in whole bacterial cell preparations.

Amidohydrolases↗
PubMedDate not supplied

Fluorescamine in cytodiagnosis of thyroid carcinomas.

Fluorescamine, which reacts with primary amino groups yielding intensely fluorescent products, was found to induce intense fluorescence in cells from medullary and papillary thyroid carcinoma. No fluorescence was detected in cells from follicular or undifferentiated carcinoma or from benign thyroid lesions, except Hürthle cell adenomas, the cells of which exhibited moderate fluorescamine-induced fluorescence. This was demonstrated on fine needle aspiration biopsy smears pretreated with formaldehyde gas. Since the fluorescamine technique is simple and rapid, it may be of value as an aid in the cytodiagnosis of thyroid neoplasms.

Biopsy, Needle↗
PubMedDate not supplied

Fluorescamine as a cytochemical detection reagent for mammary carcinoma cells.

Fluorescamine is a versatile reagent for the cytochemical demonstration of primary amino groups. Following pretreatment with formaldehyde, only certain "protected" amino groups will react with fluorescamine. In the present study we show that compounds containing such amino groups are abundant in mammary carcinoma cells but undetectable in normal mammary gland cells. Most cytologically benign breast lesions are devoid of cells containing these compounds. Although much work remains to be done, the fluorescamine technique, which is rapid and simple to perform, may offer some help in the distinction between normal and malignant cells of the mammary gland.

Breast Diseases↗
PubMed1975

Fluorescamine as a terminating agent in solid phase peptide synthesis.

Fluorescamine was shown to be an excellent terminating agent for blocking unreacted amino groups during solid phase peptide synthesis. A comparison of the termination efficiency of fluorescamine versus that of acetylation revealed that the former method gave superior products as assessed by peptide analysis, dansyl-amino end group determination and biological assay. In addition, fluorescamine terminated fragments were converted to non-fluorescent spirolactones during the deprotection stage. These spirolactones were stable to subsequent solid phase reaction conditions and were readily removed from the target peptide.

Acetylation↗
PubMed1975

Specific TLC tissue residue determination of sulfadiazine following fluorescamine derivatization.

A spectrodensitometric method for the direct determination of sulfadiazine at the tissue residue level (0.1 ppm) is based upon the measurement of the fluorescence of a sulfadiazine-fluorescamine derivative formed directly on a TLC plate by dipping it into a fluorescamine solution. The linear dynamic range for the assay is about 150 from 200 to 0.2 ng, the lower limit of sensitivity. Recoveries from various spiked tissues including milk, eggs, liver, kidneys, muscle, skin, and fat varied with the tissue type but were reproducible. The assay technique has also been used for the assay of sulfamethoxazole and has been explored for use in specifically assaying sulfonamide mixtures.

Animals↗
PubMed1997

A fluorescamine assay for membrane protein and peptide samples with non-amino-containing lipids.

A method is described for determining the concentration of membrane proteins and peptides in the presence of non-amino-containing lipids. The assay is quantitative when used for purified proteins and peptides of known sequence and qualitative when sequences are unknown or samples contain contaminating proteins. In this method, proteins and peptides are hydrolyzed to amino acids followed by derivatization by fluorescamine and spectroscopic detection in a mixed solvent system. A liquid-phase acid hydrolysis separates lipid from the sample and increases the sensitivity and accuracy of the assay. The aqueous-organic solvent, composed of 40% dimethylformamide, has two advantages. First, it suppresses differences in fluorescence between samples with and without residual hydrolyzed lipid, allowing direct comparison of samples and standards regardless of lipid content. Second, the solvent enhances the fluorescence of amino acid derivatives. While the fluorescence intensities of fluorescamine derivatives reach a maximum at approximately 40% dimethylformamide, the emission maximum wavelengths continue to blue-shift at higher concentrations of organic solvent. The selection of an acid hydrolysis mixture based on the fluorescence quenching by different acid mixtures is also reported.

Amino Acids↗
PubMed1977

Fluorescamine, a fluorescence probe for amino groups in histochemical studies of plant cells and the effect of mercury fixation.

When fixed in mercuric chloride solutions and stained with Fluorescamine, histological plant specimens emit a strong fluorescence. The fluorophore distribution is topologically identical to the staining pattern revealed by visible light methods for nucleoproteins, but the fluorescence mode of viewing preparations gave greater sensitivity and contrast than transmitted light absorption methods. The parameters that influence the formation of the fluorescent image in plant cells are discussed. The results obtained indicate that the mercury-Fluorescamine reaction is an ideal histochemical procedure for collecting qualitative and analytical information on plant nuclei and on the changes of nucleolar architecture that occur during the cellular developmental cycle.

Amines↗