SEARCH · Search PubMed

Results for “Fluorescamine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources

PubMed1983

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients.

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by any stain for primary and secondary amino groups (e.g. ninhydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in high yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.

Chromatography, Thin Layer↗

PubMed1985

A rapid HPLC technique for determining levels of histamine in tears from normal and inflamed human eyes.

Because of the important role of histamine in the inflammatory process, the measurement of histamine levels in tears has been considered as a possible index of eye irritancy. Histamine levels have been measured using an HPLC procedure involving fluorimetric detection after fluorescamine derivatization. Analyses, at picomole levels, were carried out in less than 5 minutes using 10 microliter of tear samples collected from either normal or inflamed human eyes.

Chromatography, High Pressure Liquid↗

PubMed1991

Structure of the polypeptide chain of extracellular hemoglobin from the nematode Ascaris suum.

1. Ascaris suum extracellular hemoglobin is composed of eight identical single polypeptide chain subunits carrying two heme binding sites each. 2. Limited trypsinolysis followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a major band corresponding to half the molecular mass of an intact subunit. 3. Peptide mapping of tryptic hydrolysates yielded 27 to 30 fluorescamine positive spots, about half the number of lysyl and arginyl residues in a polypeptide chain. 4. The findings indicate that a subunit of Ascaris hemoglobin consists of two structural units of roughly equal size, corresponding to two recurring sequences, connected together by the continuity of the polypeptide chain.

Animals↗

PubMed1984

An improved HPLC assay for S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) in plasma.

An HPLC assay is presented for the detection and quantitation of the radioprotective drug S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721, ethiofos) present in plasma. Improved selectivity and a 40-fold increase in sensitivity have been demonstrated over the method previously reported by this laboratory. Using precolumn derivatization with fluorescamine and S-3-(4-aminobutylamino)propyl phosphorothioate (WR-80855, a homolog of WR-2721) as the internal standard, drug levels of 0.05 to 1000 micrograms/mL were determined with excellent precision (CV less than or equal to 5% over the concentration range). An isocratic mobile phase of acetonitrile/ethanol/water (16:7:77) modified with 0.01 M tetrabutylammonium phosphate eluted the drug and the internal standard from the C-18 reverse-phase column in 23 minutes and 26 minutes, respectively. Detector response was linear over the entire range. The assay uses 150 microL of plasma and requires a total chromatography time of about 50 minutes. The method was found suitable for pharmacokinetic studies in a preliminary experiment with a beagle dog in which no interferences due to plasma constituents or drug metabolites were observed.

Amifostine↗

PubMedDate not supplied

Fluorescence histochemical methods for the study of peptide hormone-producing cells.

Fluorescence histochemical methods for the demonstration of specific residues in peptides and proteins are reviewed: Formaldehyde-ozone for NH2-terminal tryptophan, formaldehyde-HCl for tryptophan regardless of position in the peptide, OPT for NH2-terminal histidine, formaldehyde-fluorescamine for "protected" amino groups, nitroso-naphthol for tyrosine, and phenanthrenequinone for arginine residues. The methods are potent in demonstrating granule-stored material in peptide hormone-producing cells. Also quinacrine, the fluorescent anti-malaria agent, binds to granular components, as yet unidentified, in several endocrine cell types. In many cases the fluorescence histochemical methods seem to demonstrate peptides and proteins distinct from the known hormones.

Adrenocorticotropic Hormone↗

PubMed1987

Binding of formaldehyde to human and rat nasal mucus and bovine serum albumin.

The function of the nasal mucociliary apparatus, an important airway defense mechanism, is inhibited by inhaled formaldehyde. Nasal mucus, which contains significant concentrations of glycoprotein and soluble proteins, is an integral component of this system. This investigation addresses some reactions of formaldehyde with human and rat mucus in vitro in comparison with a model protein, bovine serum albumin. [14C]Formaldehyde was incubated with reconstituted preparations of human and rat nasal mucus or bovine serum albumin. Formaldehyde adducts, stabilized by sodium cyanoborohydride reduction to methylamines, were separated by Sepharose 2B gel filtration. [14C]Formaldehyde bound exclusively to one component of nasal mucus which had an elution volume identical to that of albumin. There was no detectable binding to the large molecular weight glycoproteins. The time course of reaction of formaldehyde to free amino groups was then measured using the fluorescamine technique. Formaldehyde binding was characterized by an initial fast phase (less than 2 min) followed by a slower phase which appeared to approach equilibrium (greater than 60 min). The rate of binding to human and rat nasal mucus was similar to albumin. Irreversible binding of formaldehyde to albumin was insignificant within the first 60 min indicating the reversibility of binding during this time. These data indicate that within the first 60 min, formaldehyde reacts rapidly and reversibly with nasal mucus and that it binds primarily to one component of nasal mucus. Gel filtration analysis suggests this component may be albumin although other low molecular weight proteins cannot be ruled out.

Amino Acids↗

PubMed1987

Alternative procedure for the purification of the heat-stable enterotoxin of enterotoxigenic Escherichia coli pathogenic for calves.

A method for purification of the heat-stable enterotoxin (ST) of enterotoxigenic Escherichia coli (ETEC) strains (C1444 and B41) pathogenic for calves and some physiochemical properties of the ST are described. The method involved ultrafiltration on PM-10 and UM-2 Diaflo membranes, acetone fractionation, ion-exchange chromatography on AG 1-X2, chromatofocusing and a combination of hydrophobic interaction chromatography on octyl-Sepharose CL-4B and gel-permeation on Bio-Gel P-2. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of fluorescamine-labeled purified, reduced and alkylated ST preparations revealed a single band with approximate molecular masses of 2500 and 2200 for the C1444 and B41 STs, respectively. For the C1444 ST, the final purification achieved was approximately 27,000-fold on the basis of absorbance at 280 nm per mouse effective dose. However, it was 2000-fold when calculated on the basis of mg protein per effective dose (5 ng). Amino acid composition of the C1444 ST was found to be different from that of the B41 ST suggesting that the ST produced by bovine isolates may be heterogeneous in their structure.

Animals↗

PubMed1991

Simple and rapid micro-analytical procedures for the estimation of milacemide and its metabolite glycinamide in rat plasma and cerebrospinal fluid by high-performance liquid chromatography.

A high-performance liquid chromatographic technique is described for the determination of milacemide and its primary metabolite glycinamide in rat plasma and cerebrospinal fluid. Milacemide and glycinamide are derivatized with fluorescamine to form a chromophore and a fluorophore and subsequent analysis using ultraviolet and fluorescence detectors, respectively. The extraction procedures are simple with a limit of detection 2 and 0.5 micrograms/ml for milacemide in plasma and cerebrospinal fluid, respectively, and 0.5 micrograms/ml for glycinamide in plasma or cerebrospinal fluid. The within-batch coefficients of variation for both analytes were less than 3%. Since only a small amount of sample is required, these techniques are well suited for the study of milacemide pharmacokinetics in the rat.

Acetamides↗

PubMed1995

Determination of gabapentin in serum by capillary electrophoresis.

A rapid capillary electrophoresis method for the quantification of gabapentin, a new anticonvulsant drug, in serum was developed. The assay involves derivatization of gabapentin with fluorescamine to provide a chromophore for UV-fluorescence detection. The migration time is about 11 min. The assay was linear between 0 and 20 mg/l. No other therapeutic drugs or amino acids interfered with the gabapentin peak. The relative standard deviation is 2.4% at a mean of 11 mg/l (n = 17). The mean serum level for 52 patients on this drug was 5.2 mg/l with a range of 0-12 mg/l.

Acetates↗

PubMed1988

The use of fluorescent probes in pharmaceutical analysis.

Even though many pharmaceuticals show native fluorescence, there is also an important group of compounds which is not fluorescent. A main object of studies is to make them fluorescent, principally by using fluorescent probes through derivatisation reactions. An account of the fluorescent probes more widely used for the determination of drugs and related compounds is presented in this review paper. A wide variety of fluorescent probes is described on the basis of their ability to react specifically with various functional groups. Attention is focused on derivatisation reactions used in spectrofluorimetry and chromatographic techniques (HPLC, TLC) with fluorimetric detection. The review covers only those fluorescent probes whose use involves a chemical reaction with the analyte, and not those methods that involve physico-chemical interactions such as sensitised or charge transfer processes. Many of these derivatisation reactions have been widely used in the detection of primary and secondary amines. Reagents such as dansyl chloride, fluorescamine, o-phtalaldehyde are very well known. Other reagents have also been developed for other functional groups, for example dansyl hydrazine for compounds with a carbonyl function or 4-bromomethyl-7-methoxy-coumarin for acidic compounds . Acid chlorides such as dansyl chloride may also react with different functional groups carrying active hydrogens as do phenols. The use and development of new fluorescent probes in pharmaceutical analysis is a subject for further studies.

Journal Article↗

PubMed1996

High-performance liquid chromatographic determination of taurine in human plasma using pre-column extraction and derivatization.

Plasma samples (100 microliters) were treated with 150 microliters of acetonitrile and centrifuged at 5800 g for 10 min and 50 microliters of 10 mM borate buffer (pH 9.2) were added to the supernatant solution. This was followed by the addition of a 50 microliters aliquot of 5 mM fluorescamine in acetonitrile and immediate vortex mixing. A 20 microliters sample was injected on to a reversed-phase HPLC system using a Bondclone C-18 10 microns analytical column (300 mm x 3.9 mm). The mobile phase was tetrahydrofuran-acetonitrile-phosphate buffer (15 mM, pH 3.5) (4:24:72, v/v/v). The taurine derivative was detected by measuring the UV absorbance of 385 nm. Platelet-poor plasma samples were spiked with known amounts of taurine and inter- and intra-assay calibration curves were obtained. The method was applied to the determination of taurine in platelet-rich plasma.

Chromatography, High Pressure Liquid↗

PubMed1986

The effects of myocardial ischemia and nisoldipine pretreatment on the asymmetric distribution of phosphatidylethanolamine in a canine heart sarcolemmal preparation.

We examined the distribution of phosphatidylethanolamine (PE) in the membrane bilayer of sarcolemmal preparation isolated from the ischemic and nonischemic areas of dog ventricles. The membrane preparation, isolated by the Reeves and Sutko's method, was purified ninefold over homogenates as judged from the results of measurements of (Na+K+)-ATPase and K+-p-nitrophenylphosphatase activities, sialic acid, and cholesterol. Sealed vesicles were comprised of 60% inside-out-oriented and 40% rightside-out-oriented vesicles; 30% of the total were unsealed vesicles. The results obtained from the incubation of the membrane preparation with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and cycloheptaamylose-fluorescamine complex, both of which served as nonpermeable chemical probes, indicated that 80% of the total PE was accessible from the outside. By contrast, it was possible to label up to 98% of the PE by using a permeable probe, 1-fluoro-2,4-dinitrobenzene. These results suggest that PE is predominantly localized in the cytosolic side of the sarcolemmal membrane bilayer in the dog heart. Ischemic lesion was produced in the dog heart by the occlusion of a branch of the left anterior descending coronary artery for 1.5 hr followed by 3 hr of reflow. The concentrations of both total phospholipid and phosphatidylcholine and PE in the sarcolemmal fraction prepared from the ischemic area of the myocardium were significantly decreased as compared to those from the nonischemic area. The magnitude of labeling sarcolemmal PE by TNBS was reduced in the preparation from the ischemic area as compared to that from the nonischemic area. This difference was abolished when the dog received nisoldipine (an iv injection of 5 micrograms/kg twice) or chlorpromazine (infusion at a rate of 10 micrograms/kg X min plus an iv injection of 400 micrograms/kg twice). These results suggest that ischemia decreased primarily the membrane PE existing at the cytosolic side of the sarcolemmal membrane and that pharmacological intervention can prevent the change in membrane lipids induced by ischemia.

Animals↗

PubMed1994

Cytokines in liposomes: preliminary studies with IL-1, IL-2, IL-6, GM-CSF and interferon-gamma.

A simple, generally applicable method to incorporate cytokine proteins into multilamellar liposomes is presented. A variety of human cytokines including granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins 1 alpha, 2 and 6 (IL-1 alpha, IL-2, IL-6) and interferon-gamma (IFN-gamma) were incorporated into liposomes containing a single saturated synthetic lipid, dimyristoyl phosphatidyl choline (DMPC). Sterile cytokine liposomes were produced by gamma irradiation of DMPC lipid powder prior to use in cytokine liposome synthesis. A highly sensitive and reliable fluorescamine assay to detect microgram quantities of cytokine protein associated with liposomes is also described. When a high lipid:aqueous ratio [e.g. 300 mg DMPC lipid:1.0 ml aqueous cytokine solution] was utilized, aqueous cytokines (1 mg/ml) could be incorporated with efficiencies ranging from 19% (IL-1) to > 80% (IL-2). Combinations of cytokines (e.g. IL-2 + GM-CSF) were also co-incorporated into liposomes. Experiments with IL-2, IL-6, and GM-CSF demonstrated that these cytokines remain stably associated with the DMPC lipid and do not significantly leak from liposomes when stored at 4 degrees C for at least 3 months. Washing IL-6 liposome or GM-CSF liposome preparations reliably increased the proportion of cytokine protein associated with the liposome pellet compared to free cytokine in the supernatant of centrifuged specimens. For example the proportion of GM-CSF associated with the lipid carrier increased from 34.8% (SD 2.6%) in the original preparation to 98.0% (SD 0.6%) after three washes. Differences in the pharmacokinetics of subcutaneous (sc) free GM-CSF and GM-CSF liposomes (14 mcg/mouse) were studied in BALB/c mice. Both free GM-CSF and free GM-CSF mixed with saline loaded liposomes exhibited biphasic pharmacokinetics with very high peak levels 1 and 2 h after sc injection of 14 mcg the rapid decline to very low levels after 24 h. In contrast, sc GM-CSF liposomes provided sustained and stable levels of cytokine in the serum (approximately 100 pg/ml) for 24 h. Intraperitoneal injection of GM-CSF liposomes had > 10-fold more cytokine in the peritoneal wash than free GM-CSF mixed with saline loaded liposomes. In summary, the liposome synthesis procedure described is simple and utilizes a single synthetic lipid to reliably produce sterile cytokine preparations with in vivo depot effects after either sc or ip administration. Furthermore, the method is feasible for quantities of sterile cytokine liposomes sufficient for in vivo experiments.

Animals↗

PubMed1995

Role of lysine residues in the nucleotides binding to bovine liver high-Km aldehyde reductase.

The inactivation of bovine liver high-Km aldehyde reductase (ALR) by heat (47 degrees C), 0.3 mM 2,4,6-trinitrobenzene sulfonate (TNBS) and 0.03 mM pyridoxal 5'-phosphate (PAL-P) followed pseudo-first-order kinetic as a function of incubation-time and concentration of TNBS. Nucleotides which have a 2'-phosphate group, especially beta-NADPH and beta-NADP+, showed effective protection on ALR-inactivation. However, typical substrates for ALR such as D,L-glyceraldehyde, D-erythrose, D-glucuronate and p-carboxybenzaldehyde could not protect the enzyme from inactivation. Completely inactivated enzyme was estimated to have 2.07 TNBS-modified lysine residues/mol enzyme from the determination of free amino group using fluorescamine (Ex = 390 nm, Em = 475 nm). Enzyme protected by beta-NADP+ (96.5% remaining activity) did not lose a significant number of lysine residues. Kd-values for beta-NADPH and beta-NADP+ were estimated to be 0.48 microM and 4.7 microM, respectively and TNBS-treated enzyme lost its ability to bind to these nucleotides.

Aldehyde Reductase↗

PubMed2005

The presence of oxidized phosphatidylserine on Fas-mediated apoptotic cell surface.

A growing body of evidence suggests that phosphatidylserine (PS) oxidation is linked with its transmembrane migration from the inner to the outer leaflet of the plasma membrane during apoptosis. However, there is no direct evidence for the presence of oxidized PS (PSox) on the surface of cells undergoing apoptosis. The present study was performed to detect PSox externalized to the cell surface after Fas engagement in Jurkat cells. Treatment of Jurkat cells with anti-Fas antibody induced caspase-3 activation, chromatin condensation, PS externalization, generation of reactive oxygen species, intracellular glutathione depletion, disruption of mitochondrial transmembrane potential and release of cytochrome c from mitochondria. To determine externalized PS and phosphatidylethanolamine (PE), Jurkat cells were treated with anti-Fas antibody and then labeled with membrane-impermeable fluorescamine, a probe for visualizing lipids that contain primary amino groups. Their total lipids were extracted and subjected to two-dimensional high-performance thin-layer chromatography (HPTLC). The HPTLC plate was sprayed with N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride to detect phospholipid hydroperoxides. PSox was present in small amounts within but not on the surface of normal cells. Treatment with anti-Fas antibody increased PSox within the cells and caused PSox to appear on the cell surface. In contrast, PE on the surface of Fas-ligated cells was not oxidized. Thus, the present study demonstrates for the first time the presence of PSox both within and on the surface of apoptotic cells.

Antibodies, Monoclonal↗

PubMed2005

Advanced procedures for separation and analysis of low molecular weight inhibitor (NCXIF) of the cardiac sodium-calcium exchanger.

A low molecular weight inhibitor (NCX(IF)) of the cardiac Na/Ca exchanger, isolated from the calf ventricle tissue, is capable of regulating the muscle strip's contractility and relaxation without involving the beta-activation pathway. The structural analysis of NCX(IF) requires highly purified preparations that fulfill the demanding requirements for mass spectra and NMR analyses. No such preparation is yet available. To this end, new HPLC procedures were developed by a combination of the reverse phase, normal phase, and HILIC (hydrophilic liquid chromatography) techniques. The specific activity of NCX(IF) is 10(5) times higher in the purified preparations (as compared to the crude extract) showing a 2-5% yield of total inhibitory activity and 20-100 microg content of final material. The purification yield reveals that 1 kg ventricle muscle contains 0.1-0.2 mg NCX(IF), meaning that the tissue concentrations of NCX(IF) may reach 10(-7)-10(-6) M. The diode-array scanning of purified preparations of NCX(IF) shows a homogeneous 3D peak with a maximal absorption at 202 nm. These spectral properties may represent a five-membered ring (e.g., proline, histidine) and/or simple chemical groups (like amine, carbonyl, ester, etc.), but not an aromatic ring or complex conjugates (alkyne, alkene, aldehyde, etc.). NCX(IF) does not respond to phenol/sulfur reagent, suggesting that it lacks reducing (aldo) sugar. NCX(IF) shows a faint response to fluorescamine, meaning that it may contain an amino group (or its derivative). It is believed that a combination of presently developed procedures with LC/MS and LC/MS/MS may provide a useful tool for structural analysis of NCX(IF).

Animals↗

PubMed2006

Protein bonding on biodegradable poly(L-lactide-co-caprolactone) membrane for esophageal tissue engineering.

A biodegradable and flexible poly(L-lactide-co-caprolactone) (PLLC) copolymer was synthesized and surface modification has been performed aiming at application as a scaffold in esophageal tissue engineering. The PLLC membrane surface was aminolyzed by 1,6-hexanediamine to introduce free amino groups. Using these amino groups as bridges, fibronectin and collagen were subsequently bonded with glutaraldehyde as a coupling agent. The presence of free amino groups on the aminolyzed PLLC surface was quantified using fluorescamine analysis method, which revealed that the surface NH2 density increased and eventually saturated with increasing 1,6-hexanediamine concentration or reaction time. X-ray photoelectron spectroscopy (XPS) confirmed the presence of both proteins separately on the modified PLLC surface. Water contact angle measurements evaluate the wettability of modified and unmodified PLLC surfaces. Protein-bonded surface presented more hydrophilic and homogeneous, yet PLLC can also adsorb some protein molecules. In vitro long-term (12d) culture of porcine esophageal cells proved that fibronectin- and collagen-modified PLLC surface (denoted PLLC-Fn and PLLC-Col, respectively) can more effectively support the growth of smooth muscle cells and epithelial cells; both modified and unmodified PLLC support fibroblasts growth. Mitochondrial activity assay and cell morphology observation indicate that the PLLC-Fn surface is more favorable to epithelium regeneration than PLLC-Col. These culture results provide much valuable information for our subsequent research on the construction of artificial scaffolds with esophageal function. Fibronectin-integrated PLLC will be a good candidate scaffold to support the growth of all types of esophageal cells.

Acetamides↗

PubMed2004

High-performance liquid chromatographic analysis of beta-carbolines in human scalp hair.

A chromatographic method was studied for the quantitation of beta-carbolines in hair as potent biomarkers. Under optimal conditions, human scalp hair was enzymatically digested to release analytes effectively. The hair digests were treated with fluorescamine before serial extractions to inhibit the artifactual production of beta-carbolines during analysis and purify them selectively, followed by reversed-phase high-performance liquid chromatography with fluorometric detection. Hair samples were found to contain beta-carboline and 1-methyl-beta-carboline, which were identified by tandem mass spectrometry, but not their reduced form 1,2,3,4-tetrahydro-beta-carboline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline. Both beta-carboline and 1-methyl-beta-carboline were quantified in the concentration range of 0.1-10.0 ng/ml. Their mean recoveries from hair digests were 70-72%, and the intra- and inter-assay RSD ranged between 6.0 and 10.3% in spiking experiments with standards (1.0 ng/ml). When quantitatively analyzing scalp hair collected from alcoholics, smokers, non-smokers and autistics, beta-carboline and 1-methyl-beta-carboline showed the concentrations of ng/mg levels or less which characterized different hair samples. The proposed method will be useful for detecting the in vivo concentration changes of beta-carbolines associated with alcohol abuse, smoking behavior and neuropsychiatric disorder.

Alcoholism↗