Chemiluminescence activated by rhodamine G in plasma in the presence of divalent iron ions

Rhodamine G was found to activate blood plasma chemiluminescence induced by ferrous ions. The dye in concentrations 300-500 mole/l increased chemiluminescence by an order of magnitude. The luminescence was inhibited by histidine and SOD. A conclusion may be drawn that the mechanism of the activated ferrous chemiluminescence with rhodamine G was related to superoxide anion-radicals and singlet oxygen.     

The inhibition of peroxyradical-induced chemiluminescence by melanins

Model melanins synthesized from adrenochrome, DOPA and pyrocatechol quench the peroxyradical-coupled chemiluminescence initiated by electrolysis, photolysis and redox reactions in several selected systems. Calculations of the selfadsorption (inner filter effect) of melanins that decreases chemiluminescence intensity as well as changes in absorption spectra of photolabile substrate-probes such as cytochrome c and rhodamine, lead to the conclusion that melanins scavenge peroxyradicals and inhibit chemiluminescence. The inhibition effect (beta-value) is much higher than that of most powerful antioxidants in all chemiluminescence systems excluding the luminol-hydrogen peroxide one. Donor-acceptor properties of melanins, pertinent to their antioxidizing activity, are discussed.     

Enhanced chemiluminescence for trazodone trace analysis based on acidic permanganate oxidation in concurrent presence of rhodamine 6G

A new sensitized chemiluminescence method by acidic permanganate oxidation was developed for the sensitive determination of trazodone. A fluorescent dye as used rhodamine 6G to increase a chemiluminescence intensity. Under optimum conditions, the liner range of the calibration curve was obtained for 1–5000 nmol/L. The limit of detection was calculated from 3σ of a blank was 0.23 nmol/L. The coexistent ions and substances had no interference with the chemiluminescence measurement. The chemiluminescence spectra were measured to elucidate a possible mechanism for the system. The present method was satisfactorily used in the determination of the drugs in pharmaceutical samples and animal serums.     

Determination of cardamonin using a chemiluminescent flow-injection method

A sensitive and selective flow injection chemiluminescence method for the determination of cardamonin over the range 1.0 x 10(-8) to 8.0 x 10(-6) g/mL is described. The method is based on the enhancement by cardamonin of the chemiluminescence of the reaction between cerium (IV) and rhodamine 6G in sulphuric acid medium. The optimised flow injection procedure yielded a detection limit for cardamonin of 8.8 x 10(-9) g/mL, whilst the relative standard deviations of intraday and inter-day precision were below 2.5%. The method has the advantages of high sensitivity and a wide linear range. It was successfully applied to the determination of cardamonin in Alpinia katsumadai Hayata. The mechanism of the chemiluminescence reaction is proposed.     

Isolation and characterization of an apoprotein from the d less than 1.006 lipoproteins of human and canine lymph homologous with the rat A-IV apoprotein.

The singlet oxygen traps, 2,5-diphenylfurane and 1,3-diphenylisobenzofurane were oxidized to cis-benzoylethylene and o-dibenzoylbenzene during the decomposition of diisopropyl-N-nitrosamine catalyzed by peroxidase. Singlet oxygen quenchers inhibited this conversion and also the chemiluminescence accompaying the catalyzed reaction. The chemiluminescence is enhanced by 1,4-diazobicyclo (2.2.2) octane, fluorescein, eosin rhodamine B and rose bengal but little effect was detected in the presence of 9,10-dibromoanthracene-2-sulfonate, 9,10-diphenylanthracene-2-sulfonate and anthracene-2-sulfonate. An emission spectrum of the unsensitized reaction in 560 – 600 nm region was observed. It is concluded that singlet oxygen is formed during peroxidase catalyzed degradation of diisopropyl-N-nitrosamine.     

A novel chemiluminescence method for the determination of orciprenaline based on ferricyanide-rhodamine 6G.

A novel flow injection chemiluminescence method for the determination of orciprenaline was developed. The method is based on the chemiluminescence (CL) reaction of orciprenaline with potassium ferricyanide in sodium hydroxide medium, sensitized by the fluorescent dye rhodamine 6G. The proposed procedure allows quantitation of orciprenaline in the concentration range 0.01-1.2 microg/mL, with a detection limit of 7.2 x 10(-3) microg/mL. The relative standard deviation (RSD) is 2.7% for 0.1 microg/mL orciprenaline (n = 9). The sampling frequency was calculated at approximately 120/h. The method was successfully applied to the determination of orciprenaline in pharmaceutical preparations. A brief discussion on the possible CL reaction mechanism is presented.     

Effect of He-Ne laser radiation on the chemiluminescence of mouse spleen cells

A study was made of He-Ne laser radiation (lambda = 632.8 nm) on spontaneous chemiluminescence of mouse splenic cells and that stimulated by addition of Candida albicans. Irradiation with low-intensity red light was shown to stimulate cell chemiluminescence and to intensify that stimulated by C. albicans within the dose range from 100 to 300 J/m2 with a maximum at about 200 J/m2.     

Rhodamine 6G inhibits the matrix-catalyzed processing of precursors of rat-liver mitochondrial proteins

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the alpha and beta subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B.D. (1984) Eur. J. Biochem. 144, 387-392].     

Flow injection chemiluminescence determination of dihydralazine sulphate based on permanganate oxidation sensitized by rhodamine B.

A novel flow injection chemiluminescence (CL) method for the determination of dihydralazine sulphate (DHZS) is described. The method is based on the CL produced during the oxidation of DHZS by acidic permanganate solution in the presence of rhodamine B. Rhodamine B is suggested as a fluorescing compound for the energy-transferred excitation. The CL emission allows quantitation of DHZS concentration in the range 5-800 ng/mL, with a detection limit of 1.9 ng/mL (3sigma). The experimental conditions for the CL reaction are optimized and the possible reaction mechanism is discussed. The method has been applied to the determination of DHZS in pharmaceutical preparations and compares well with the high performance liquid chromatography (HPLC) method.     

Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides

The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations. The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM). The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C). The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM). Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition. Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition. With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete. The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined. Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation on the interaction between dihydroxybenzene and Fe3+- H2O2-Rh6G system based on enhancing chemiluminescence.

A highly sensitive flow-injection chemiluminescence (FI-CL) method has been developed for the determination of dihydroxybenzene, based on the hydroxyl radical reaction. Hydroxyl radical (.OH) produced by the reaction of Fe(3+) and H(2)O(2) oxidize rhodamine 6G to produce weak CL. It was observed that catechol and hydroquinone greatly enhanced the weak CL reaction. However, the proposed CL system is not suitable for determination of resorcinol because the enhancement reaction is very slow. The proposed procedure has a linear range of 0.01-2 mg/L for catechol, with a detection limit of 0.006 mg/L, and 0.008-1 mg/L for hydroquinone, with a detection limit of 0.004 mg/L. The possible mechanism of the CL system is discussed.     

Development of a compact capillary electrophoresis–chemiluminescence system with ultra‐fast peroxyoxalate reaction to monitor the hydrolysis of rhodamine 6G

A simple and effective capillary electrophoresis–chemiluminescence (CE–CL) detection system was developed based on an ultra‐fast bis(2,4,6‐trichlorophenyl)oxalate (TCPO) chemiluminescence (CL) reaction (0.6 s duration) that avoided overlapping peaks and peak tailing. Through a series of static injection experiments, this unusually rapid CL reaction was ascribed to the catalytic effect of imidazole in the tetrahydrofuran solvent, which has been rarely utilized in such investigations. A possible mechanism is given to explain the results. Under the optimized conditions, rhodamine 6 G (R6G) and its hydrolysis product (R6G‐COOH) could be efficiently separated through electrophoresis in 7 min, with sensitive CL detection in the proposed CE–CL system. In this way, the alkaline hydrolysis of R6G was monitored, followed by estimation of relative rate constants and activation energy. This finding and application should be helpful in further study for the TCPO CL reaction, and revealed an attractive opportunity for simplifying the CE–CL system, such as in a microchip device. Copyright © 2011 John Wiley & Sons, Ltd.     

Ascorbate- and hemoglobin-dependent brain chemiluminescence

It has been indicated recently that ascorbic acid is responsible for the hemoglobin-mediated oxidative damage to the central nervous system (Sadrzadeh & Eaton, J. Clin. Invest. 82:1510-1515, 1988). In this paper we describe the changes in chemiluminescence accompanying hemoglobin- and ascorbate-dependent oxidative injury to brain tissue. Addition of either hemoglobin (15 microM) or ascorbate (1 or 2 mM) to rat brain homogenates stimulated spontaneous chemiluminescence in a synergistic manner. This increase in chemiluminescence was inhibited by desferrioxamine indicating that free iron was involved in the reactions leading to lipid peroxidation. Preincubation with ascorbate oxidase inhibited both spontaneous and hemoglobin-dependent chemiluminescence, suggesting that ascorbate was required for the reactions leading to lipid peroxidation. Supplementation with aminotriazole (an irreversible inhibitor of the catalase-H2O2 complex) increased chemiluminescence in a time-dependent manner, as catalase reacted with accumulated H2O2, suggesting that ascorbic acid has a dual action being involved in the production of H2O2 and also maintaining Fe in the reduced state to catalyze a Fenton-like reaction. The excited species responsible for the chemiluminescence were partially characterized by adding specific fluorescent energy acceptors: dibromoanthracene (DBA) and diphenylanthracene (DPA). Both DBA and DPA stimulated chemiluminescence several-fold indicating that triplet and singlet species are responsible for the observed chemiluminescence. Excited singlet carbonyls (identified with DPA) may be produced during the collision of two ROO.. Singlet oxygen may also be generated during the same reaction. It decays to the triplet state (emitting chemiluminescence at 634 nm) and reacts with double bonds producing dioxetanes, which may breakdown generating triplet carbonyls (identified with DBA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of tsumacide residues in vegetable samples using a flow-injection chemiluminescence method.

A sensitive, simple and rapid flow-injection chemiluminescence (FI-CL) method is described to determine tsumacide pesticide residue based on the CL reaction of the alkaline degradation product of tsumacide with acidic KMnO(4) when rhodamine 6G was present. Under the optimum conditions, the relative CL intensity is linear with the concentration of tsumacide in the range of 2.0 x 10(-3)-0.20 mg/L. The detection limit is 6.6 x 10(-4) mg/L (3sigma) and the relative standard deviation for 2.0 x 10(-2) mg/L tsumacide solution was 2.28% (intra-day) and 4.85% (inter-day). The proposed method has been applied to determine the residue of tsumacide in vegetable samples and the recovery test is very satisfactory.     

Chemiluminescence of synephrine based on the cerium(IV)-rhodamine B system.

A new chemiluminescence (CL) method is described for the determination of synephrine. It is based on the reaction between synephrine and Ce(IV) in a nitric acid medium and measurement of the CL intensity produced by rhodamine B used as a luminophore, similar to luminol or lucigenin in basic media, instead of as a sensitizer. In the optimum conditions, the increase of CL intensity was correlated with synephrine concentration over the range 5.0 x 10(-9)-1.0 x 10(-6) g/mL with a detection limit of 1.0 x 10(-9) g/mL. The relative standard deviation (RSD) was 2.9% for 1.0 x 10(-7) g/mL synephrine (n = 11). The method was applied to the determination of a drug in herbal products, citrus fruit and biological samples, with satisfactory results. The results given by the proposed method are in good agreement with those given by HPLC-UV and UV spectrophotometry.     

The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.     

Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

Oxygenation of anaerobically isolated brain and liver homogenates is associated with chemiluminescence and formation of lipid hydroperoxides, the latter determined by the thiobarbituric acid assay. Light emission and formation of malonaldehyde are 20-fold higher in the brain than in liver; chemiluminescence of both decays when accumulation of malonaldehyde ceases. Exogenous organic peroxides, such as t-butyl hydroperoxide, inhibit the light-emission response to oxygenation by brain homogenate, whereas they enhance that of liver homogenate. t-Butyl hydroperoxide-induced photoemission of liver homogenate shows a polyphasic kinetic pattern that is O2-dependent. The spectral analysis of chemiluminescence arising from brain and liver homogenates on oxygenation shows a spectrum with five emission bands at 420-450, 475-485, 510-540, 560-580 and 625-640 nm. These bands are subjected to intensity changes or shifts of the wavelength whenever t-butyl hydroperoxide is present, either inhibiting or stimulating light emission. The blue-band chemiluminescence, around 435 nm, is possibly due to the weak light emission arising from excited carbonyl compounds [Lloyd (1965) J. Chem. Soc. Faraday Trans. 61, 2182-2193; Vassil'ev (1965) Opt. Spectrosc. (USSR) 18, 131-135], whereas the presence of other bands suggests generation of singlet molecular oxygen either in the process triggered on oxygenation (lipid oxygenation) or after supplementation with organic hydroperoxides. We offer several explanations for the spectral analysis presented here.     

Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone. Evidence for a third drug-binding site.

P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport.     

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.     

Catalysis of singlet oxygen production in the reaction of hydrogen peroxide and hypochlorous acid by 1,4-diazabicyclo[2.2.2]octane (DABCO)

The kinetics of the singlet oxygen production in the hydrogen peroxide plus hypochlorous acid reaction were studied by measuring the time course of the singlet oxygen emission at 1268 nm. The addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) increased the peak intensity of the chemiluminescence, but decreased its duration. The increased rate of singlet oxygen production likely accounts for the enhancement of singlet oxygen dimol emission reported in 1976 by Deneke and Krinsky (J. Am. Chem. Soc. 98, 3041-3042). This phenomenon was not seen when singlet oxygen was generated with the reaction of hypobromous acid and hydrogen peroxide. Thus, the enhancement of red chemiluminescence by DABCO should not be regarded as a general test for the production of singlet oxygen in complex biochemical systems.