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At least 307 records · Page 17Linked to original sources
PubMed1977

Characteristics of influex and net flux of amino acids in Mytilus californianus.

1. Intact mussels and an in vitro preparation of isolated gill tissue were employed to study characteristics of the influx and net flux of amino acids in Mytilus californianus. 2. The kinetics of influx of 14C-labelled amino acids were complicated by the presence of a rapidly labelled extracellular compartment. 3. Correction of influx data for the extracellular compartment revealed influx of 14C-cycloleucine and 14C-glycine to be mediated by a transport mechanism adequately described by Michaelis-Menten kinetics. Passive diffusion plays no significant role in influx. 4. Influx and net flux of glycine into intact Mytilus were examined. From high concentrations (greater than 20 micronM) the influx of 14C-glycine was equivalent to the net influx of primary amine as determined by fluorescamine. At low ambient concentrations (greater than 20 micronM), influx of 14C-glycine occurred more rapidly than net influx as determined by fluorescamine. The data suggest that influx of labelled substrate is accompanied by efflux of unknown primary amine(s). In the absence of labelled substrate, efflux continues until a steady-state concentration of 2-5 micronM in the medium is achieved.5. The rate of influx of 14C-cycloleucine into isolated gill tissue, and the concentration gradient which can be developed by gill tissue are reduced when sodium is replaced in the medium. 6. The efflux of primary amines from isolated gill tissue is stimulated by the replacement of sodium in the medium with choline. 7. The data are consistent with a sodium-coupled mechanism for the transport of amino acid into gill tissue. However, energetic considerations cast doubt on the adequacy of such a mechanism to account fully for the observed trans-epidermal transport.

Amino Acids↗
PubMed2005

[Determination of chloramphenicol residues in milk by reversed-phase high performance liquid chromatography with fluorescence detection].

A high performance liquid chromatographic method with fluorescence detection for the determination of chloramphenicol (CAP) residues in milk was developed. Although CAP itself is non-fluorescent the aromatic nitro group of CAP could be reduced to aromatic primary amino group and fluorescamine can be used as a selective reagent for primary amines. Therefore, the CAP was derivatized with fluorescamine prior to injection in this project. The HPLC method was performed on a Diamond C18 column (250 mm x 4.6 mm i. d., 4.0 microm) with the mobile phase composed of sodium acetate buffer (0.02 mol/L, pH 6.0) -acetonitrile-tetrahydrofuran (76: 16: 8, v/v/v). The flow rate of the mobile phase was set at 1.0 mL/min, and the column was maintained at 40 degrees C. Analytes were detected by a fluorescence detector at 410 nm excitation and 508 nm emission wavelength. The standard curve was linear in the range from 0.4 microg/L to 800 microg/L. The limit of detection (LOD) was 0.2 microg/L, and the limit of quantitation (LOQ) was 0.4 microg/L. Overall recoveries were between 66.6% and 92.8% with relative standard deviations between 4.5% and 9.4%. The procedure provides a rapid, reliable and sensitive method for the determination of CAP in milk.

Animals↗
PubMed2000

Derivatization, extraction and concentration of amino acids and peptides by using organic/aqueous phases in capillary electrophoresis with fluorescence detection.

We report a novel method that facilitates sample pretreatment and detection in amino acid analysis by coupling solvent extraction with capillary electrophoresis. Amino acids and peptides were fluorescently labeled, concentrated into an organic solvent, and then separated by capillary zone electrophoresis with fluorescence detection. To achieve this, acetophenone was first employed to dissolve the derivatizing reagent, fluorescamine. The products, which possessed both hydrophilic and hydrophobic moieties, could be extracted and concentrated into the organic phase by suppressing the deprotonation of carboxyl groups, thus enhancing the hydrophobicity of the resulting molecules through pH modification in the aqueous solution. Furthermore, by fine-tuning the pH value, individual amino acids and short peptide molecules could be separated selectively from the sample bulk. This convenient, chemically controllable concentration technique may be useful in sample concentration and purification of biologically related samples such as amino acids and short peptides.

Amino Acids↗
PubMed2002

Performance of an ultraviolet light-emitting diode-induced fluorescence detector in capillary electrophoresis.

The performance of a fluorescence detector in capillary electrophoresis (CE) using a light-emitting diode (LED) as excitation source is reported. An ultraviolet LED pulsed at a repetition rate of 500 Hz, combined with a time-discrimination and averaging acquisition system, was used. Limits of detection of 3 and 18 fmoles (at a signal-to-noise ratio equal to 3) were achieved for fluorescamine-derivatized bradykinin and lysine, respectively. This system exhibited a linear response for a concentration range between 54 and 417 microM for derivatized lysine, and between 1.81 and 23.58 microM for derivatized bradykinin. This detection system showed to be very convenient for routine analytical applications.

Aspartic Acid↗
PubMed1978

Protein sorption on polymer surfaces measured by fluorescence labels.

Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about 10(-8) g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.

Adsorption↗
PubMed1989

Asymmetric distribution of phospholipids in the membrane of vesicles released during in vitro maturation of guinea pig reticulocytes: evidence precluding a role for "aminophospholipid translocase".

Guinea pig reticulocytes lose their transferrin (Tf) binding activity during maturation, in the form of vesicles (exosomes) released into the extracellular medium. Vesicles were prepared from cultures of reticulocytes to study the possible externalization of a particular membrane-associated activity, i.e., that of "aminophospholipid translocase." Analysis of the peptide composition of these vesicles revealed that the major proteins are the Tf receptor and another peptide (70kDa), which is probably the "clathrin-uncoating ATPase" described by Johnstone et al. (1987). The exosome had a lipid composition similar to erythrocyte membrane, although with a lightly but significantly lower phosphatidylethanolamine content. The aminophospholipid distribution in the vesicle membrane was determined by fluorescamine labeling. The exosomes showed an asymmetric aminophospholipid distribution similar to that of erythrocytes. "Aminophospholipid translocase" activity was absent, as no transverse diffusion of spin-labeled phospholipids occurred over more than 2 hours at 37 degrees C.

Animals↗
PubMed1975

Rapid fluorometric determination of procainamide hydrochloride dosage forms.

A fluorometric procedure for procainamide hydrochloride was developed, and it offers improvements in ease, speed, and sensitivity over the official method. The new procedure is based on the reaction with fluorescamine in aqueous medium at pH 7.5 to form a fluorophore, with activation and emission wavelengths of 400 and 485 nm, respectively. The fluorescence is linear (r = 0.999) over the 0. 04-1 mug/ml concentration range and is stable for at least 2 hr. Recovery data appeared to be accurate, quantitative, and reproducible. The overall recovery was 99.8% with a standard deviation of +/-1.14 (n = 5). The method was successfully applied to commercially available dosage forms.

Capsules↗
PubMed1977

Biological disposition of rigid analogs of amphetamine.

Tissue levels of amphetamine and of amphetamine analogs with a rigid conformation (2-aminoindan, 2-aminotetralin, and 2-aminobenzocycloheptene) were measured in rats by a spectrofluorometric method involving the reaction of the primary amine group with fluorescamine. All drugs were concentrated in tissues, the order of distribution being lungs greater than kidneys greater than liver=spleen=brain greater than muscle greater than fat=heart greater than blood. In brain, amphetamine and 2-aminoindan were present mostly in the supernatant fraction after high speed centrifugation onbrain homogenates; the two higher molecular weight drugs were present at slightly greater concentrations in the particulate fraction. All four drugs disappeared from rat brain with half-lives of 1-2 hr. Iprindole pretreatment increased drug levels in brain and prolonged the half-lives by two- to threefold. The data suggest that the biological disposition of the rigid analogs resembles generally that of amphetamine and that all of the drugs are probably metabolized by ring hydroxilation in the rat.

Amphetamines↗
PubMed1994

Quantitation of urinary 1,2,3,4-tetrahydro-beta-carboline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed to quantify 1,2,3,4-tetrahydro-beta-carboline (TBC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) in human urine. Urine samples with added internal standard were subjected to a reaction with fluorescamine and solvent extractions to remove the precursor tryptamine, which readily condenses with aldehydes in samples and reagents. Such a pretreatment completely suppressed the artifactual formation of TBC and MTBC during analytical procedures. The purified original tetrahydro-beta-carbolines and the internal standard were separated by reversed-phase ion-pair chromatography with fluorescent detection. Their simultaneous separation was automatically completed in a short time (< 12 min). Both TBC and MTBC were quantified at ng/mL concentrations. The quantitative results revealed a wide variation in urinary levels of TBC and MTBC, possibly indicating that their considerable amounts excreted in the urine originate from dietary sources.

Adolescent↗
PubMed1994

A nonradioactive fluorescent gel-shift assay for the analysis of protein phosphatase and kinase activities toward protein-specific peptide substrates.

Synthetic peptides are important tools with which to study the activities of protein kinases and phosphatases toward specific substrate sequences which are present within selected regions of a protein. Most existing assays for the phosphorylation or dephosphorylation of such peptides utilize 32P and either affinity chromatography or HPLC separation and require extensive characterization and validation. Here, we describe a method for monitoring the phosphorylation or dephosphorylation of almost any peptide of interest which does not require the use of radioactivity, making its reagents stable for a prolonged period, and which can be performed in any standard laboratory. For this, after performance of kinase or phosphatase reactions with the peptide of interest, products are derivatized with fluorescamine and are separated according to charge by agarose gel electrophoresis. Phosphorylated and nonphosphorylated peptides are readily separated and can be both identified and quantified by uv detection. The lower limit for detection of peptide in the agarose gel was 0.02 nmol using the gel-shift kinase assay with cAMP-dependent kinase and Kemptide as substrate. This had sensitivity and reproducibility similar to those of a standard assay using [gamma-32P]ATP with this substrate. Dephosphorylation of a synthetic phosphopeptide corresponding to a segment of the cholecystokinin receptor was tested in an analogous assay with known amounts of protein phosphatase 2A. Phosphopeptide and dephosphopeptide were easily detected and quantified with as little as 0.03 mU/ml protein phosphatase 2A activity. Therefore, with this assay, most synthetic peptides and phosphopeptides can be used as substrates without further modification. This will be of particular interest for monitoring the purification of highly specific protein kinase and phosphatase activities.

Adenosine Triphosphate↗
PubMed1994

Simultaneous measurement of cytochrome P4501A catalytic activity and total protein concentration with a fluorescence plate reader.

A method for simultaneous measurement of the cytochrome P4501A-associated enzyme, ethoxyresorufin-O-deethylase (EROD), and total protein concentration in liver microsomes is described. EROD assays were carried out in multiwell plates, and the fluorescent product (resorufin) and total proteins were quantified within the same wells with a fluorescence plate reader. Fluorescamine was used for protein assays.

Animals↗
PubMed2001

A fluorescence-based homogeneous assay for measuring activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is one of the key enzymes of bacterial lipid A biosynthesis, catalyzing the removal of the N-acetyl group of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The lpxC gene is essential in Gram-negative bacteria but absent from mammalian genomes, making it an attractive target for antibacterial drug discovery. Current assay methods for LpxC are not suitable for high throughput screening, since they require multiple product separation steps and the use of radioactively labeled material that is difficult to prepare. A homogeneous fluorescence-based assay was developed that uses UDP-3-O-(N-hexyl-propionamide)-N-acetylglucosamine as a surrogate substrate. This surrogate can be prepared from commercially available UDP-GlcNAc by enzymatic conversion to UDP-MurNAc, which is then chemically coupled to n-hexylamine. Following the LpxC reaction, the free amine of the deacetylation product can be derivatized by fluorescamine, thus generating a fluorescent signal. This surrogate substrate has a K(m) of 367 microM and k(cat) of 0.36 s(-1), compared to 2 microM and 1.5 s(-1) for the natural substrate. Since no separation is needed, the assay is easily adaptable to high throughput screening. IC(50)s of LpxC inhibitors determined using this assay method is similar to those measured by traditional method with the natural substrate.

Amidohydrolases↗
PubMed1983

Radiolabeled DNase, a potential indicator for noninvasive detection of tissue damage.

Pancreatic DNase I was labeled with 131I or fluorescamine and injected IV into NMRI mice bearing a sarcoma 180. Of the injected tracer, 1.5%-2% was found to accumulate per g tumor. In sections of tumor tissue DNase was localized in damaged cells in solid and necrotic tumor regions. This binding is most probably due to specific interaction of DNase with actin, an ubiquitous cytoskeletal protein. Two-component blood clearance with a rapid first component (two-thirds of applied radioactivity) was observed. The labeled tumor could easily be visualized by gamma camera imaging. The findings suggest DNase to be a potent radiopharmaceutical for imaging damaged tissue, occurring in malignant tumors as well as in infarcts, and inflammatory lesions.

Animals↗
PubMed1983

Microfluorometric studies on chromosomes. Quantitative determination of protein content of Chinese hamster chromosome 1 in situ with and without trypsin digestion.

The fluorescent stains, dansyl chloride and fluorescamine, were used to indicate the amount of protein in G-banded and unbanded chromosomes 1 of the Chinese hamster relative to the average amount of protein in human erythrocyte. Fluorescence was found to be proportional to protein mass up to the equivalent of three erythrocytes. In G-banding, trypsin digestion resulted in an average protein loss of 35.4% compared with unbanded chromosomes.

Animals↗
PubMed1977

Extended application of flow microfluorometry by means of dual laser excitation.

A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region, DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specificity combined with a great stability under UV illumination. By separating two focussed laser beams at their intersecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.

DNA↗
PubMed1982

Cellular and subcellular localization of polyamines cytochemical methods providing new clues to polyamine function in normal and neoplastic cells.

Studies on the cellular and subcellular localization of polyamines have previously been hampered by the rapid redistribution of these organic cations during cell fractionation. Cytochemical methods selectively localizing polyamines would, hence, be important for our understanding of where polyamines are stored and where they work. With the aid of chemical characterizations of tissue extracts, biosynthesis experiments and cytochemical model experiments we show that the cytochemical formaldehyde-fluorescamine (FF) technique selectively detects the polyamines spermine and spermidine. Results obtained with the FF method concur with previous chemical observations by showing that growing and neoplastic cell populations are rich in cytochemically detectable polyamines. The FF method also localizes polyamines to certain normal secretory cell systems, including pancreatic exocrine and endocrine (insulin) cells, cells and secretion products in the ventral prostate, pituitary growth hormone cells and neurosecretory nerve endings and leucocytes. In most cell systems studied, polyamines appear to be most concentrated in cytoplasmic structures. However, in HeLa cell mitotic chromosomes and in frog red cell nuclei most polyamines seem to be chromosomes and in frog red cell nuclei most polyamines seem to be associated with chromatin. These observations raise important questions concerning both extra- and intracellular functions of polyamines.

Animals↗
PubMed1989

Human erythrocyte damage at the initial stages of oxidative stress.

Specific fluorescent probes have been used to monitor changes in erythrocyte membranes in the first stages of the hemolytic process induced by irradiation with visible light in the presence of protoporphyrin IX. Although no change, or even a slight increase of fluorescence anisotropy, occurred with two probes having a preferential binding to membrane proteins, such as fluorescamine and 3-pyrene maleimide, the fluorescence anisotropy of two lipophilic probes, namely diphenyl-hexatriene and anilino-naphthalene sulfonate, underwent a substantial decrease upon irradiation. Concomitantly, a dramatic decrease of ATPase activity and an increase of thiobarbituric-reacting substances were observed in erythrocyte membranes. Instead, there was no effect on the activities of the intracellular enzymes glucose-6-phosphate dehydrogenase and pyruvate kinase. These findings are consistent with the hypothesis that protoporphyrin-sensitized irradiation induces, primarily in the erythrocyte membrane, the peroxidation of the lipid component, which results in an increase of the fluidity of the bilayer. Hemolysis eventually occurs because of an osmotic imbalance resulting from the combination of increased passive diffusion and decreased active ion transport.

Cytoplasm↗
PubMed1983

An enzymatic fluorometric assay for L-lysine in plasma and tissue.

A simple, specific, and reliable method has been developed for the determination of L-lysine in blood plasma and tissue. The L-lysine in the sample is decarboxylated enzymatically, and fluorescamine is added to a pentan-1-ol extract of the cadaverine formed. This produces a stable product which is measured fluorometrically.

Cadaverine↗