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At least 451 records · Page 25Linked to original sources
PubMed1985

High-performance liquid chromatographic determination of plasma and brain histamine without previous purification of biological samples: cation-exchange chromatography coupled with post-column derivatization fluorometry.

A highly sensitive and specific method was developed for the determination of histamine in biological materials by high-performance liquid chromatography with a cation exchanger and an automated Shore's fluorometric detection system. Since substances causing interference in Shore's o-phthalaldehyde method, such as ammonia, histidine, spermine and spermidine, were completely separated on the column and their fluorescent intensities were much less than that of histamine in this detection system, histamine could be determined by injecting a perchloric acid extract of human plasma or mouse brain tissue directly onto the column without any previous purification procedure. The lower limit of detection of histamine by this method is 0.05 pmol, and the within-day and day-to-day variations in plasma histamine assay are less than 3%. The plasma histamine level in normal human subjects was found to be 4.0 +/- 1.6 pmol/ml (mean +/- S.D., n = 20). A good linear correlation was obtained between values for the histamine contents of mouse brain tissues determined by this method and by a radioenzymatic method with a purified histamine-N-methyltransferase preparation. The histamine levels of whole brain, hypothalamus, thalamus, brain stem and frontal cortex in male ddY mice were 367 +/- 38, 1143 +/- 70, 414 +/- 66, 196 +/- 47 and 467 +/- 91 pmol/g of wet tissue (mean +/- S.D.), respectively.

Adult↗
PubMed1999

Simultaneous high-performance liquid chromatographic determination of retinol by fluorometry and of tocopherol by ultraviolet absorbance in the serum of newborns.

The simultaneous determination of retinol and tocopherol by isocratic HPLC in 100 microl serum from preterm newborns is described. Retinol (tR 2.02+/-0.04 min) and retinyl acetate were detected fluorometrically, and were baseline-resolved in 4 min. Tocopherol (tR 8.4+/-0.16 min) and tocopheryl acetate were detected by UV absorbance. Intra- and inter-assay RSD were: retinol, 5.6 and 8.1, and tocopherol, 3.6 and 6.7, respectively. This method is fast, selective and highly sensitive for retinol. It permits the measurement of serum concentrations of retinol and tocopherol with good accuracy and precision.

Chromatography, High Pressure Liquid↗
PubMed2000

Measurement of atmospheric hydrogen peroxide and hydroxymethyl hydroperoxide with a diffusion scrubber and light emitting diode-liquid core waveguide-based fluorometry

We describe a new automated instrument for measuring gas- and aqueous-phase H2O2. The chemistry relies on the hematin-catalyzed oxidation of nonfluorescent thiamine to fluorescent thiochrome by H2O2; this reaction is 25-fold more selective for hydrogen peroxide than its nearest alkyl hydroperoxide congener, CH3HO2. The optical characteristics of the fluorescent product are such that it is ideally excited by newly available GaN-based light emitting diodes emitting in the near-UV. A stable long-life miniature flow-through fluorescence detector based on a transversely illuminated liquid core waveguide is thus used for this purpose. The limit of detection (LOD, S/N = 3) for liquid-phase H2O2 is 11 nM. A temperature-controlled high-efficiency Nafion membrane diffusion scrubber is used to collect gaseous hydrogen peroxide with near-quantitative efficiency with an S/N = 3 LOD of 13.5 pptv. The system responds to hydrogen peroxide and hydroxymethyl hydroperoxide but not to methyl hydroperoxide. The use of very inexpensive and stable reagents, highly sensitive detection, benign chemistry, and a fluorescence detector using a solid-state illumination source results in a particularly affordable automated instrument. Design and performance details and illustrative results from a 1999 field campaign (Atlanta Supersite Study) are presented.

Journal Article↗
PubMed1995

Indirect detection of aromatic hydrocarbons by semiconductor laser fluorometry in micellar electrokinetic chromatography.

A semiconductor laser is used as an exciting light source in indirect fluorescence detection of neutral samples separated by micellar electrokinetic chromatography. A surfactant, tetradecyltrimethylammonium chloride, is used to form micelles, which act as a pseudostationary phase in the chromatographic separation. This surfactant has a positive charge, thus preventing adsorption of a visualizing agent, oxazine 750 (which contains a positive charge), to the capillary wall (which is negatively charged). The detection mechanism is based on the exclusion of the fluorophore, which is located in the hydrophilic part of the micelle, by a hydrophilic sample. The fluorescence intensity is reduced when the fluorophore is released into the aqueous solution. Separations of several aromatic compounds are demonstrated, and the parameters affecting the selectivity are discussed.

Chromatography↗
PubMed2001

Time-resolved total internal reflection fluorometry study on polarity at a liquid/liquid interface.

The polarity of a water/oil (oil: cyclohexane, carbon tetrachloride, toluene, chlorobenzene, o-dichlorobenzene, or 1,2-dichloroethane) interface was investigated by means of time-resolved total-internal-reflection (TIR) fluorescence spectroscopy of a polarity-sensitive probe: sulforhodamine B (SRB). In bulk solutions, the nonradiative decay rate constant of SRB increased with an increase in a solvent polarity parameter [ET(30)], and this relationship was used to estimate the polarities of water/oil interfaces. For the oil having a relatively low solvent polarity [ET(30) < 35 kcal/mol], the polarity of the water/ oil interface agreed with that of the arithmetic average of the polarities of the two phases [ET(30)calc]. For water/odichlorobenzene and water/1,2-dichloroethane interfaces [ET(30) of the oil > 35 kcal/mol], on the other hand, the interfacial polarity determined by TIR spectroscopy was lower than the ET(30)calc. The results are discussed in terms of thickness/roughness of the water/oil interface.

Journal Article↗
PubMed2003

Pulsed excitation source multiplexed fluorometry for the simultaneous measurement of multiple analytes. Continuous measurement of atmospheric hydrogen peroxide and methyl hydroperoxide.

Presently, solid-state sources such as light-emitting diodes (LEDs) provide for intense, nearly monochromatic light. They are available over a broad range of emission wavelengths. Unlike incandescent and discharge lamps, LEDs can be turned on and off at high speeds. The resulting light pulses are highly reproducible. This allows the use of a single photomultiplier tube (PMT), often the most expensive component in a high-sensitivity measurement system, as a multiplexed detector with multiple, fiber-optic-coupled, fluorescence-detection cells excited by solid-state sources. A time resolution of 1 min is adequate in many continuous detection schemes. This enables multiple-channel single-detector multiplexed measurement without any loss of S/N. On the basis of this principle, we describe a new automated continuous instrument for the simultaneous measurement of atmospheric hydrogen peroxide and methyl hydroperoxide (MHP). A Nafion membrane diffusion scrubber (DS) is used with hematin-catalyzed oxidation of thiamine to thiochrome for the measurement of H2O2, and an expanded poly(tetrafluoroethylene) (ePTFE) DS is used with a H2O2 destruction catalyst and horseradish peroxidase-catalyzed oxidation of thiamine to thiochrome for the measurement of MHP. The respective limits of detection are 25 pptv and 15 pptv. Design, performance details, and illustrative results from a field campaign (Philadelphia NEO3PS study, 2001) are presented.

Journal Article↗
PubMed2003

Direct observation of molecular recognition mediated by triple hydrogen bonds at a water/oil interface: time-resolved total internal reflection fluorometry study.

Molecular recognition mediated by hydrogen-bonding interactions at a water/CCl(4) interface was investigated directly by means of time-resolved total internal reflection (TIR) fluorescence spectroscopy. The TIR fluorescence decay profile of riboflavin (RF) in the absence of a guest in the CCl(4) phase was fitted satisfactorily by a single-exponential function. In the presence of N,N-dioctadecyl-[1,3,5]triazine-2,4,6-triamine (DTT) as a guest in the CCl(4) phase, on the other hand, the fluorescence decay profiles were best fitted by double-exponential functions with the relevant amplitude (A(i)) being varied with the concentration of DTT. Furthermore, the rotational reorientation time of RF at the interface determined by fluorescence dynamic anisotropy was 210 ps in the absence of DTT, while fast (160-220 ps) and slow (670-750 ps) rotational reorientation times were observed in the presence of DTT. This slow rotational reorientation time was shown to ascribe to that of the RF-DDT complex formed at the water/CCl(4) interface. These results indicate that molecular recognition mediated by complementary hydrogen bonding takes place effectively at the water/CCl(4) interface, which was observed directly by both fluorescence dynamics and fluorescence dynamic anisotropy measurements under the TIR conditions.

Journal Article↗
PubMed1992

Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants.

A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Förster [Förster, T. (1948) Ann. Phys. (Leipzig) 2, 55-75].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry.

Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.

Benz(a)Anthracenes↗
PubMed1988

Nanosecond pulse fluorometry of conformational change in phenylalanine hydroxylase associated with activation.

Conformational change in rat liver phenylalanine hydroxylase associated with activation by phenylalanine or N-(1-anilinonaphth-4-yl)maleimide was investigated by measuring fluorescence spectra and fluorescence lifetimes of tryptophanyl residues as well as the probe fluorophore conjugated with SH groups of the hydroxylase. The fluorescence spectrum of tryptophan exhibited its maximum at 342 nm. It shifted by 8 nm toward longer wavelength accompanied by an increase in its intensity, by preincubation with 1 mM phenylalanine. The fluorescence intensity of tryptophan increased by 36% upon the activation. On the other hand, the binding of (6R)-L-erythro-tetrahydrobiopterin, a natural cofactor of the enzyme, induced a decrease in the fluorescence intensity by 79% without a shift of the maximum wavelength. The fluorescence lifetime of tryptophan of phenylalanine hydroxylase exhibited two components with lifetimes of 1.7 and 4.1 ns. The values of the lifetimes changed to 1.4 and 5.6 ns, respectively, upon the activation. It is considered that the change in the longer lifetime is correlated with the shift of the emission peak upon the activation. The values of both the lifetimes decreased to 0.64 and 3.6 ns upon the binding of (6R)-L-erythro-tetrahydrobiopterin, which is coincident with the decrease in the fluorescence intensity. Conjugation of N-(1-anilinonaphth-4-yl)maleimide with SH of phenylalanine hydroxylase brought about a decrease in both the fluorescence intensity and the value of the shorter lifetime of the tryptophanyl residues, while the longer lifetime remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Multiple conformational states in myoglobin revealed by frequency domain fluorometry.

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Interactions of a new antitumor antibiotic BBM-928A with deoxyribonucleic acid. Bifunctional intercalative binding studied by fluorometry and viscometry.

A new actinoleukin-like antitumor antibiotic, BBM-928A, has been shown to interact with isolated DNA molecules. BBM-928A contains two substituted quinolines linked by a cyclic decapeptide. Quenching effects of the covalently closed superhelical PM2 DNA on the BBM-928A fluorescence revealed a strong interaction with an apparent association constant of 1.93 x 10(7) M-1 and with 11 deoxyribonucleic acid (DNA) nucleotides per BBM-928A binding site. Viscometric studies indicated the BBM-928A induced an unwinding-rewinding process of the closed superhelical PM2 DNA typically observed for DNA intercalators. The unwinding angle (43 degrees) induced by BBM-928A was almost twice that of the ethidium bromide (26 degrees), a monofunctional intercalator. The BBM-928A-induced increase of the helix length of sonicated rodlike calf thymus DNA was approximately 1.5-fold that induced by the ethidium bromide. On the basis of these observations, we concluded that BBM-928A bifunctionally intercalated with DNA in a manner similar to the bifunctional intercalation of echinomycin.

Antibiotics, Antineoplastic↗
PubMedDate not supplied

Monitoring cell concentration and activity by multiple excitation fluorometry.

Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.

Candida↗