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PubMed1997

Site-specific fluorescence labeling of the beta2 adrenergic receptor amino terminus.

A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications: (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.

Adenylyl Cyclases↗

PubMed2001

Use of a fluorescence microplate reader for the detection and characterization of metal-assisted peptide hydrolysis.

Metal ions and complexes that hydrolyze peptides under nondenaturing conditions of temperature and pH hold great promise for use in protein structural studies. However, the extreme stability of the peptide amide bond has placed limits on the number of reagents available. In addition, the development of new cleavage strategies has been hindered by the fact that no facile procedure exists for the detection and characterization of metal-assisted peptide hydrolysis. Here we describe a rapid assay in which a microplate reader is used to detect fluorescence produced by the reaction of fluorescamine with hydrolyzed peptides. We have employed this assay to detect Zn(II) and Pd(II)-assisted peptide hydrolysis in multiple samples and in each case have extended our approach to a successful analysis of reaction kinetics. Aliquots from multiple time points are treated with fluorescamine in a single 96-well plate. Because the plate is scanned in a microplate reader in only 58 s, the assay is very convenient compared to conventional approaches which rely on NMR and HPLC to monitor individual reactions. Using our assay, rate constants and half-lives are easily derived from the kinetic data by means of linear regression curve fits of triplicate runs.

Automation↗

PubMed1990

Roles of proteins in cation/membrane interactions of isolated rat cardiac sarcolemmal vesicles.

To ascertain the roles of the membrane proteins in cation/sarcolemmal membrane binding, isolated rat cardiac sarcolemmal vesicles were extensively treated with Protease (S. aureus strain V.8). SDS-gel electrophoresis, protein and phosphate analysis confirmed that at least 20-22% of the protein, but none of the phospholipid, was solubilized by this procedure, and that the remaining membrane proteins were extensively hydrolyzed into small fragments. The cation binding properties of the treated vesicles were then examined by analyzing their aggregation behavior. The results demonstrate that this procedure had no effect on the selectivity series for di- and trivalent cation binding, or the divalent cation-induced aggregation behavior of the sarcolemmal vesicles at different pHs, indicating that proteins are probably not involved in these interactions and cannot be the low affinity cation binding sites previously observed. It did, however, change the pH at which protons induced sarcolemmal vesicle aggregation, suggesting a possible role for proteins in these processes. Protease treatment also modified the effects of fluorescamine labelling on divalent cation-induced vesicle aggregation, indicating that the NH2 groups being labelled with fluorescamine are located on the sarcolemmal proteins. Together, these results support the hypothesis that di- and trivalent cation binding to the sarcolemmal membrane is largely determined by lipid/lipid and/or lipid/carbohydrate interactions within the plane of the sarcolemmal membrane, and that membrane proteins may exert an influence on these interactions, but only under very specialized conditions.

Animals↗

PubMed2004

Interactions of Abeta(1-40) with glycerophosphocholine and intact erythrocyte membranes: fluorescence and circular dichroism studies.

Deposition of amyloid beta peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimer's disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid beta(1-40) peptide (Abeta) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of Abeta and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled Abeta studies indicate GPC enhances Abeta aggregation. CD spectroscopy reveals that Abeta in the presence of GPC adopts 14% more beta-sheet structure than does Abeta alone. Fluorescamine anisotropy measurements show that GPC and Abeta interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble Abeta and GPC insert into the phospholipid head-group region of the membrane where they interact leading to beta-sheet formation in soluble Abeta which enhances Abeta aggregation.

Alzheimer Disease↗

PubMed1985

Influences of metal ions on the reaction of amino and imino acids with fluorogenic reagents.

When a twofold excess of metal ions (Cu2+, Ni2+, Zn2+, Mg2+, Ca2+, Fe3+, and Al3+) was added to a reaction medium of amino acids (1-50 microM each) with the fluorogenic reagents NBD-F (4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole), DNS-Cl (5-N,N-dimethylaminonaphthalenesulfonyl chloride), OPA (o-phthalaldehyde), and fluorescamine (4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione), a reduction of derivatization was observed. The effect was especially marked on the reaction with NBD-F. The derivatization of His and Asp with DNS-Cl and fluorescamine and of His with OPA was suppressed by Cu2+ and/or Ni2+. The suppression by the metal ions can be overcome by the addition of EDTA to the reaction medium. For determination of a small amount of amino and imino acids (less than microM) the addition of a chelating reagent to biological samples is recommended before derivatization with these electrophilic reagents.

4-Chloro-7-nitrobenzofurazan↗

PubMed1978

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor o.

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.

Cell Membrane↗

PubMed1986

Does diamide treatment of intact human erythrocytes cause a loss of phospholipid asymmetry?

Diamide-treated human erythrocytes have been compared with native red cells as to the accessibility of their amino phospholipids to both phospholipase A2 hydrolysis and fluorescamine labeling. In agreement with observations by others (Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Biochim. Biophys. Acta 509, 21-32), treatment of intact human erythrocytes with diamide resulted in considerably enhanced degradation of amino phospholipids upon subsequent incubation of the cells with bee venom phospholipase A2. The hydrolysis of phosphatidylethanolamine (PE) in control cells reached a plateau value at 5% after 10 min. In diamide-treated cells, on the other hand, PE hydrolysis did not level off. Contrastingly, dose-response curves recorded for the labeling of PE with the very fast reacting NH2-group-specific reagent, fluorescamine, showed identical results for both native and diamide-treated erythrocytes. In each of these two cases, a plateau was reached after approx. 15% of the PE had been labeled. These results strongly suggest that the enhanced phospholipase-A2-induced hydrolysis of amino phospholipids in diamide-treated erythrocytes may reflect a destabilization of the lipid bilayer, rather than an in situ loss of phospholipid asymmetry.

Azo Compounds↗

PubMed1987

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum.

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca2+-ATPase forms monomer-type (P1) type crystals in the E1 and dimer-type (P2) crystals in the E2 conformation. The P1 type crystals are induced by Ca2+ or lanthanides; P2 type crystals are observed in Ca2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca2+-ATPase in the E2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5'-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.

Animals↗

PubMed1987

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection.

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.

Androgen-Binding Protein↗

PubMed1982

Fluorescence probes specific for the plasma membrane of intact human erythrocytes and lymphocytes.

Human erythrocytes and lymphocytes were isolated from venous blood and subjected to one of two protocols. In one protocol the suspended cells were labeled with fluorophore (fluorescamine or 12(9)AS). This procedure was followed sequentially by cellular lysis, cellular fractionation, and fluorescence and absorption readings. In the other protocol the suspended cells were lysed, and then the cellular homogenate labeled with fluorophore followed by cellular fractionation and spectroscopy readings. The lymphocytes were fractionated into plasma membrane, cytosol, and nuclear-mitochondrial fractions and the erythrocytes into plasma membrane and cytosol fractions. The results demonstrate that under the given labeling conditions, both fluorescamine and 12(9)AS are highly localized to the plasma membrane of intact human erythrocytes and lymphocytes. Furthermore, by P-31 NMR analysis, fluorophore labeling did not alter cellular high energy phosphate metabolism or cellular permeability to Mn2+. Therefore, these fluorophores are potentially powerful probes of human erythrocyte and lymphocyte plasma membrane dynamics in inherited and acquired disease states.

Cell Membrane↗

PubMed1992

Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test.

Mutagenicity testing of biological samples and proteins is complicated by the presence of histidine and histidine-related growth factors which may produce a false positive result in the Ames/Salmonella plate incorporation test. A bioassay method, utilizing an automated dispenser-photometer and Salmonella typhimurium strain TA1535 as the indicator bacteria, was used to estimate the presence of histidine-related growth factors in three enzyme solutions submitted for mutagenicity testing. One of the solutions was clearly positive in the Ames/Salmonella test and also contained the highest amount of L-histidine-HCl-equivalents. The two other solutions, with low or undetectable amounts of L-histidine-HCl-equivalents, gave equivocal and negative results, respectively, in the Ames/Salmonella test. Studies were also performed with strains TA98, TA100 and TA1535 to determine the amount of added L-histidine-HCl that would result in a 'positive' result in the Ames/Salmonella test. Because the minimum amount of L-histidine-HCl required to double the number of revertant colonies was 150 nmol/plate, and the maximum amount of L-histidine-HCl-equivalents supplied by the enzyme preparations was 40 nmol/plate at the highest tested dose, the mutagenicity test results of the enzyme solutions cannot be explained solely by histidine or related compounds. Smokers' and non-smokers' urines, concentrated with liquid extraction (CHCl3) and adsorbent (XAD-2 and XAD-2/Sep-Pak C18) techniques, were studied to reveal differences in efficiencies to extract histidine and histidine-related compounds in the urines. Amounts of 'histidine' in concentrates of urine were measured using the bioassay method and a chemical method employing derivatization with fluorescamine. The fluorescamine method also efficiently detected 3-methyl-L-histidine, a product of muscle metabolism excreted in urine, which was found to be unable to support auxotrophic growth in TA1535, leading to exaggerated estimations of the auxotrophic growth enhancing properties of urine extracts. The urine extracts, and pure L-histidine-HCl, were tested using a two-step fluctuation test to estimate auxotrophic growth factor effects in this type of test. Because of a strong dilution effect when adding the histidine-free selection medium, the fluctuation test employed in this study was not found to be particularly sensitive to growth factors. The results of this study indicate that use of a bioassay, employing the same indicator bacteria as the mutagenicity test themselves, is a reliable way to measure histidine-related growth factors in biological samples.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzymes↗

PubMed1988

The use of fluorescent probes as markers for sheep lymphocyte subpopulations.

Sheep blood lymphocytes were labelled with fluorescent probes and examined under the fluorescence microscope and by the fluorescence-activated cell sorter. A novel probe using fluorescamine, coupled to hexylamine, detected 22.9% of cells, apparently of the B-cell series, counted by fluorescence microscopy. Substitution of fluorescein isothiocyanate (FITC) for the fluorescamine did not label the same subpopulation of cells although the lymphocytes could then be examined in the cell sorter. A larger number of cells (38.8%) formed the brighter cluster but did not behave as B cell when separated on nylon-wool columns. Improvement in discrimination of the cell populations was obtained with FITC-hexadecylamine (C16). This probe detected 38% of cells in the smallest cluster, 44% of cells in the intermediate cluster and 19% of cells in the brightest cluster. The proportion of cells in each cluster appeared to parallel closely the "null", erythrocyte (E) rosette-forming T cells and the B cells detected by conventional markers for blood lymphocytes. Other fluorescent probes, formed from FITC and other amines and amino acids, labelled lymphocyte membranes. Probes with a terminal charge labelled the small cluster particularly well, whereas those that were terminally non-polar labelled the larger cells brigthly, but not to the same intensity as the charged probes in the small cells.

Animals↗

PubMed1976

A comparative study on bovine alpha-lactalbumin and lysozyme by nanosecond fluorometry.

Analysis of the time decay of fluorescence anisotropy of 1-dimethylaminoaphthalene-5-sulfonyl (DNS) and fluorescamine derivatives of bovine alpha-lactalbumin and lysozyme reveals that no significant differences in mean rotational relaxation times are present. While fluorescamine molecules appear to orient randomly on these proteins, DNS is bound with a preferential orientation. Other fluorescence characteristics of the labels are also cited.

Animals↗

PubMed1995

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides.

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective RF values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.

Animals↗

PubMed1984

High-performance liquid chromatographic detection and quantitation of amines in must and wine.

Three fluorigenic reagents were tried in order to increase the sensitivity of the detection of various amines. The derivatives formed were then used to develop a reversed-phase high-performance liquid chromatographic (HPLC) procedure for the separation of at least five amines. Dns-C1 and fluorescamine were rejected. The chromatogram of Dns-amines from red wine was overcrowded with unidentifiable peaks. It was then postulated that ammonia or phenol derivatives or other by-products of the Dns derivatization reaction interfered with the separation of amines. Fluorescamine, although it produced highly fluorescent derivatives, had the drawback of reacting with di- and polyamines to give more than one derivative and this interfered with the resolution. o-Phthaldialdehyde (OPT) was used successfully for the derivatization of amines in red must and wine. The method involved the reaction of amines with OPT in the presence of mercaptoethanol followed by extraction of the derivatives with ethyl acetate. A reversed-phase HPLC system was developed for the separation of OPT derivatives of agmatine, cadaverine, ethanolamine, histamine, phenylethylamine, putrescine, tryptamine, tyramine, spermine and spermidine within 40 min.

Amines↗

PubMed1980

The use of a pre-column for the direct high-performance liquid chromatographic determination of the anti-depressants clovoxamine and fluvoxamine in plasma.

A high-performance liquid chromatographic system for the direct determination of the antidepressants clovoxamine and fluvoxamine in plasma is described. The primary amines are derivatized with the fluorogenic reagent fluorescamine in order to increase the sensitivity and selectivity, but also to decrease the polarity of the amines. The band broadening of some combinations of pre-column and analytical column is compared for the fluorescamine derivative of fluvoxamine. The combination of a pre-column containing RP-2 and an analytical column containing RP-8 has been successfully used for the determination of the two antidepressants in plasma. At relatively low concentrations of the drugs, a simple step-gradient elution is required for the removal of a large proportion of the more polar components of the samples. Concentrations in the range of 10-1000 ng clovoxamine per ml plasma were determined by means of external standardization and show a good correlation with the data of a more laborious gas chromatographic method. The detection limits for clovoxamine and fluvoxamine are approximately 3 ng/ml in plasma.

Antidepressive Agents↗

PubMedDate not supplied

Bilayer distribution of phosphatidylserine and phosphatidylethanolamine in lipid vesicles.

The distribution of phosphatidylethanolamine (PE) and phosphatydilserine (PS) in liposomes was studied as a function of aminophospholipid concentration using fluorescamine as labeling reagent. The method is suitable for such determination since, in the assay conditions, fluorescamine does not penetrate the vesicles nor does it disrupt them. The liposomes were obtained by sonication, extrusion, or mechanical dispersion (MLV). For any kind of vesicle, the percentage of PS in the external monolayer is higher than that obtained for PE in the corresponding vesicles. In extruded PS liposomes, this aminophospholipid is located preferentially in the outer layer, while for PE liposomes the localization depends on the size of vesicle. Sonicated liposomes present an asymmetrical distribution of both aminophospholipids, and the external location of PS or PE always predominates. In contrast, in MLV, aminophospholipids are mainly found in the inner layers of the vesicles, except for liposomes formed by the lowest PS proportion. A remarkable feature of PS liposomes is the reduction of vesicle size, especially in MLV liposomes, in comparison with neutral liposomes.

Fluorescamine↗

PubMed1977

Quantitative determination of transformed cells in a mixed population by stimultaneous fluorescence analysis of cell surface and DNA an individual cells.

Cell-surface labeling with fluorescamine indicates that the fluorescence of Balb 3T3 A31 cells in considerably decreased after both viral and chemical transformation. This phenomenon, coupled with the technique of flow microfluorometry, enabled nontransformed and transformed cells to be distinguished. A second fluroescent probe, propidium iodide, which intercalates into DNA, was used in combination with fluorescamine in order to obtain a ratio of cell-surface labeling to DNA content. This manipulation allowed enhanced resolution of two populations and the detection of small numbers of transformants in a predominantly normal population.

Cell Line↗