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At least 19 recordsLinked to original sources
PubMed1976

Studies on fluorescamine: Part I--Applications of fluorescamine in forensic toxicological analysis.

This paper describes some applications of the fluorescamine spot test to forensic toxicological analysis. The fluorescamine test only reacts with primary amines; thus, this test makes a clear-cut distinction between amphetamine and methamphetamine. Previous common spot tests used reacted the same with these two amines. Fluorescamine is 100 times more sensitive in detecting amphetamine extracted from urine on thin-layer chromatograms than ninhydrin. Thus, it is a more sensitive method of detecting amphetamine abuse in urinalysis screening programs.

Amphetamine↗
PubMed1975

Characterization of an angiotensin II-fluorescamine derivative.

The coupling of fluorescamine (4-phenylspiro[furan-2(3H), 1'-PHTHALAN]-3,3'dione) to angiotensin II to form a fluorescent derivative was studied. Complete reaction of the peptide below concentrations of 10- minus 4 M could be achieved with a fluorescamine concentration of 0-3 mg ml- minus 1 of acetone at pH 8-3, and the lowest concentration detectable by fluorescence spectroscopy was 100 pmol ml- minus 1. The derivative, as prepared did not react with ninhydrin, and no fluorescence was generated when fluorescamine was reacted with (1-Sar)-ATII. These data suggest that fluorescence is generated only through the coupling of fluorescamine to the N-terminal primary amine of ATII. The ATII-fluorescamine derivative has the same intrinsic activity on the contraction of rat colon (elevenfold loss of affinity), and on the release of fluorogenic corticosteroids from bovine adrenal cortical slices (sixfold loss of affinity) compared to ATII. Water-hydrolysed fluorescamine and Asp-fluorescamine did not contract rat colon preparations; the contractile response to ATII-fluorescamine was blocked by (8-Leu)-ATII, a specific ATII antagonist. These findings suggest that theATII fluorophore shares a common receptor site with the native octapeptide. The rate loss of biological activity of the ATII-fluorescamine derivative was appreciably lower than that observed for ATII. The present study suggests that the ATII-fluorescamine derivative can be substituted for radioactively-labelled ATII for use in a variety of applications.

Adrenal Cortex↗
PubMedDate not supplied

[Nuclear ribonucleoproteins containing pro-mRNA. XIV. Structural study using ethidium and fluorescamine].

Nuclear 30S RNP particles were studied by means of fluorescence techniques. It's shown that fluorescamin interacts with NH2-groups of protein molecule. As a result, covalent fluorescent label is formed. Quantum yield (rho), fluorescence spectra, lifetime of excited state (tau) and polarization of fluorescamin complexes with 30S particles were studied. Excitation spectra have their maximum at 395 nm, and fluorescence spectrum at 480 nm. These figures correspond to spectra of fluorescamin complexes with NH2-groups of lysine. Mean quantum yield (rho = 0.27) and lifetime of excited state of fluorescence (tau = 7.8 nsec) were measured. It's shown that fluorescamin forms two types of fluorescent complexes in 30S particles. These complexes differ only by their rho(rho1 = 0.11, rho2 = 0.30) and rho(rho1 = 3.6 nsec, rho2 = 10.0 nsec) by 2.7 times. Migration radius between fluorescamin bound to protein and ethydium bromide adsorbed on double-stranded regions of pre-mRNA in RNP-particles was measured. It's equal to 32 A. Adsorbtion isotherms of ethydium bromide were measured by fluorescence in 0.1 and 0.4 M NaCl. Data obtained showed that 6% of pre-mRNA in 30S particles bound the dye as a strong complex, i. e. this part of pre-mRNA is double-stranded. RNase treatment of RNP had no effect on this value. But the increase of NaCl concentration up to 0.4 M caused the dissociation of protein subunits to some extent followed by appearance of up to 40% free NH2-groups interacting with fluorescamin. Measuring of energy migration from fluorescamin to ethydium bromide showed that double-stranded pre-mRNA regions strictly bound to protein sticked out from RNP particle at a distance of about 27 A. The increase of NaCl concentration up to 0.4 M leads to disruption of this strict bond of double-stranded regions with protein. As a result, these regions of pre-mRNA become labile and move away from the RNP particle at more than 30 A. According to theoretical calculations, there is about 1--2 pre-mRNA hairpins (18--9 base pairs respectively) per one 30S particle.

Ethidium↗
PubMed1989

The use of fluorescamine as a detection reagent in protein microcharacterization.

With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.

Amino Acid Sequence↗
PubMed1979

Formaldehyde-fluorescamine-induced fluorescence as a property of carcinoma cells.

Fluorescamine is a sensitive cytochemical probe for primary amino groups and produces an intense general fluorescence in unfixed tissue sections reflecting the ubiquitous occurrence of such groups. Following treatment with formaldehyde, most primary amino groups react to form derivatives unable to yield fluorescence with fluorescamine. Certain cell systems, however, contain amino groups which do not react with formaldehyde but display strong reactivity with fluorescamine. In formaldehyde- and fluorescamine-treated specimens such cell systems display an intense fluorescence, whereas the majority of tissue constituents are non-fluorescent. Fluorescent cell systems include certain protein- and peptide-secreting cells and a large number of different types of carcinoma cells. In some cases it appears that neoplastic transformation is necessary before the cells display formaldehyde-fluorescamine-induced fluorescence. Available data indicate that the reactive substance(s) are peptide in nature and that the production of such substance(s) may be a general property of carcinoma cells.

Carcinoma↗
PubMed1977

The functional and fluorescence properties of Escherichia coli RNA polymerase reacted with fluorescamine.

1. Fluorescamine (4-phenylspiro[furan-2,(3)1'-phthalan]-3,3'-dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration. Excess fluorescamine is rapidly hydrolysed. Reaction is with xi-amino gruops of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning. 2. The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes. The relative decrease of fluorescence is greatest in the betabeta' subunits. Holoenzyme and core enzyme show essentially the same behavior. 3. The inactivation of activity by fluorescamine is primarily at the level of initiation. Template binding and chain propagation are less affected. 4. The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm. The fluorescence lifetime is in the range of 3-8 ns and the emission is highly polarized. In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template. 5. Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound. The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine. An average transfer distance of approx. 4-5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template.

Binding Sites↗
PubMed1975

Fluorescamine as a histochemical reagent: demonstration of polypeptide hormone-secreting cells.

Fluorescamine is a useful fluorescence microscopic reagent for the demonstration of certain peptide hormone-secreting cells in formaldehyde-fixed tissues. Among the cells demonstrated are the pituitary GH cells, the gastrin cells, the insulin cells and the thyroid C cells. In the latter cell system degranulation brings about a marked decrease in the intensity of the fluorescamine-induced fluorescence suggesting that the fluorogenic component is associated with the secretory granules. In models neither of the hormones tested--GH, gastrin, insulin--gave fluorescamine-induced fluorescence after pretreatment with formaldehyde. It is suggested that the fluorescamine-induced fluorescence of the endocrine cells demonstrates granular components distinct from the hormones.

Animals↗
PubMed1977

Labeling of membranes from erythrocytes and corn with fluorescamine.

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.

Cell Fractionation↗
PubMed1976

Derivatization of compounds at the origin of thin-layer plates with fluorescamine.

A method has been developed for the detection of compounds with primary amino groups on thin-layer chromatography (TLC) plates with fluorescamine. The compounds dissolved in buffer were applied to TLC plates and derivatized at the origin by developing with or dipping into an acetone-hexane solution of fluorescamine. Virtually all fluorescamine derivatives stayed at the origin, and they were subsequently separated using appropriate solvent systems. As little as 10 pmoles of fluorescamine-derivatized compound could be detected when viewed under a long-wave (366 nm) ultraviolet lamp. The method was applied to the analysis of peptides, amino acids and amines.

Amines↗
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↗
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↗
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↗
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↗
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↗