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At least 145 records · Page 8Linked to original sources
PubMed1983

Improved liquid chromatographic determination of serum cortisol with double internal standardization compared to radioimmunoassay and fluorometry, and evaluated by isotope dilution/mass spectrometry.

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the determination of cortisol in only 200 microliters of serum is described. Cortisol and two internal standards, 19-nortestosterone (IS1) and 6 alpha-methylprednisolone (IS2) are extracted with dichloromethane and analyzed on a C18 reversed-phase column eluted with a mobile phase of methanol:water at a flow rate of 0.75 ml/min. Ultraviolet absorption at 254 nm is used for detection and quantitation is performed by peak height ratio measurement. Using 200 microliters of serum, the lower limit of detection for cortisol is 10 ng/ml, the analytical recovery is 104 +/- 3.6% (n = 8), and the day-to-day precision was 1.69% at a level of 90 ng/ml (n = 16). Cortisol values obtained by this method were generally lower than those obtained by radioimmunoassay or by fluorometry. A serum pool was analyzed both by HPLC and by isotope dilution/mass spectrometry (ID/MS). A mean value of 90.1 ng/ml was obtained by HPLC (n = 16, CV = 1.7%), whereas ID/MS yielded a mean of 90.8 ng/ml (n = 28, CV = 0.4%). These results clearly demonstrate the high specificity and the accuracy of the HPLC procedure. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of medication by either of these two products.

Chromatography, High Pressure Liquid↗
PubMed1987

Correction for contaminant fluorescence in frequency-domain fluorometry.

We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.

Anthracenes↗
PubMed1988

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry.

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.

Calcium↗
PubMed1989

Nanosecond fluorometry of the single tryptophan in cytochrome P-450e (P450IIB2).

Properties of the single tryptophan residue in rat liver microsomal phenobarbital-inducible cytochrome P-450e (P450IIB2) were studied by the nanosecond time-resolved fluorometry. The tryptophan fluorescence decay time was found to be 3.6 ns and it was not affected by the addition of substrate (perhydrophenanthrene). This result strongly indicates that the tryptophan residue is not a part of the substrate-binding site.

Animals↗
PubMed1989

Steady-state fluorescence anisotropy and multifrequency phase fluorometry on oxidized phosphatidylcholine vesicles.

Multilamellar liposomes, from mixtures of unoxidized (control) and singlet oxygen oxidized phosphatidylcholine, were studied by steady-state fluorescence anisotropy and multifrequency phase fluorometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Lifetime fluorescence decay of the DPH-labeled liposomes was analyzed either by a model of discrete exponential components and a model that assumes a continuous distribution of lifetime values. Increasing the oxidized phosphatidylcholine content in the liposomes, an increase of the membrane interior polarity and a decrease of membrane fluidity occurs which can be related to the hydroperoxide-lipids and double bonds conjugation, respectively.

Diphenylhexatriene↗
PubMed1994

Determination of trace amounts of albumin in human bronchoalveolar lavage fluids by fluorometry with chromazurol S.

Fluorometry using chromazurole S (CAS) was applied to determine trace amounts of albumin in human bronchoalveolar lavage fluids (BALF). The calibration curve was linear in the range of 5-60 micrograms/ml of albumin. The CAS method was proven to be much more selective for albumin than for IgG. Freezing of BALF samples did not affect albumin analysis by the CAS method after storage at -20 degrees C for 80 days. This finding suggests that albumin in the BALF samples is stable under these conditions. The correlation was highly linear (r = 0.966) between the albumin levels in concentrated BALF samples (n = 47) determined by the CAS method and by radial immunodiffusion. The CAS method is sensitive enough to determine albumin levels in unconcentrated BALF samples, whereas radial immunodiffusion often requires concentration. The former method is more suitable for measuring albumin in BALF samples than the latter, because concentration by ultrafiltration results in poor reproducibility. The concentration of albumin in BALF samples of healthy volunteers (n = 5) and patients with sarcoidosis (n = 32) was determined by the CAS method. There was a statistically significant difference (P < 0.01) in the albumin levels in BALF samples between healthy subjects and patients with sarcoidosis at a clinically active state (n = 15). This finding shows that the determination of albumin levels in BALF samples is useful for investigating lung diseases and that the CAS method is promising in the determination of trace albumin in BALF samples, because it is simple, sensitive and precise.

Bronchoalveolar Lavage Fluid↗
PubMed1983

Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, non-invasive fluorometry and physiological correlates.

The pyridine nucleotides from both the epithelium and the endothelium of rabbit cornea were measured by the cycling assay. Sodium azide (10 mM) applied for 1 hr to induce histotoxic anoxia decreased the endothelial NAD+/NADH ratio from 4.62 to 1.49 and decreased the epithelial NAD+/NADH ratio from 2.56 to 1.08. The larger NAD+/NADH ratio for the endothelium as compared to the epithelium corresponds to a more oxidized state. The corresponding ratios for NADP+/NADPH were 1.2 for the endothelium and 0.70 for the epithelium. Sodium azide had no effect on the NADP+/NADPH ratio for the endothelium, but decreased the epithelial ratio to 0.62. Pyridine nucleotide fluorescence was measured with a difference corneal fluorometer on the perfused whole cornea preparation and the perfused everted corneal preparation. Sodium azide (10 mM) for 30 min resulted in a 19.4 +/- 0.7% increase in the pyridine nucleotide fluorescence from the whole corneal preparation and a 4.5 +/- 0.6% increase from the everted endothelial preparation. Corneal anoxia induced by stopping the perfusion on the endothelial side resulted in a 18.7 +/- 0.6% increase in pyridine nucleotide fluorescence for the whole corneal preparation. Sodium azide (10 mM) resulted in a 35% decrease in the transendothelial potential difference and a 76% decrease in the rate of transendothelial fluid transport. A comparison is made between invasive chemical analysis and real time, non-invasive fluorometry to measure histotoxic corneal anoxia.

Animals↗
PubMed1975

Nanosecond pulse fluorometry in polarized light of dansyl-L-cysteine linked to a unique SH group of F-actin; the influence of regulatory proteins and myosin moiety.

The order of magnitude of the correlation time, which characterizes the dansyl cysteine residue linked to F-actin is ten times greater than the correlation time of the G-actin monomer [1]. Still it is much smaller than the correlation times of the F-actin polymer as a whole. The dansyl chromophore reveals that the C terminal end of the actin peptide chain, is mobile. As Ebashi and his co-workers have shown (13), Ca2+ triggers muscular contraction by acting on F-actin through the mediation of the regulatory proteins troponin and tropomyosin. By using spin label technique, Tonomura et al. [14] found that Ca2+ induces a conformational change on the troponin, tropomyosin actin complex. The quasi elastic scattering of laser light measurement of Fujime and Ishiwata [15] showed that troponin-tropomyosin F-actin has a rotational correlation time in the millisecond range which characterizes the flexibility of this complex; Ca2+ induces an increase of this flexibility. The present pulse fluorometry study shows an increase of mobility of the fluorescent probe induced by Ca2+. It seems difficult to correlate the results of the two kinds of measurements as long as we do not know the exact nature of the fluorescent kinetics unit.

Actins↗
PubMed1985

Early transmembrane events in tumour cell responses observed by stopped-flow fluorometry.

Early transmembrane events of tumour cells (mouse myeloma X5563 and lymphoma RDM4) after binding of a monoclonal antibody against mouse MHC antigen and a mitogenic lectin, Con A, were examined by stopped-flow fluorometry with 3 different fluorescent probes. The results showed that membrane fluidities of the cells increased first after binding of anti H-2Kk monoclonal antibody (11-4.1), then calcium was released from intracellular stores into the cytoplasma, and lastly calcium influx occurred from the external medium into the cytoplasma. While Con A only induced calcium influx from the external medium into the cytoplasma.

Animals↗
PubMed1981

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry.

The measurement of fluorescence lifetimes is known to be hindered by the wavelength-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize these effects in photomultiplier tubes used for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelengths of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatterer decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelength. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from -55 to +55 degrees C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference lifetime, to calculate the actual phase and modulation of the excitation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of NADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.

Indicators and Reagents↗
PubMed1986

Picosecond resolution of oxytocin tyrosyl fluorescence by 2 GHz frequency-domain fluorometry.

The technique of frequency-domain fluorometry has been extended to 2000 MHz using the harmonic content of a picosecond laser source and a microchannel plate photomultiplier tube. This new instrument was used to resolve complex subnanosecond intensity and anisotropy decays of the tyrosyl emission of oxytocin. The intensity decay was found to contain at least three exponential components, 80, 359 and 927 ps. The anisotropy analysis revealed a 29 ps torsional motion of the tyrosine residue as well as a 454 ps overall rotational correlation time. The time resolution of this method should permit the comparison of experimental results with theoretical models for motions of proteins.

Kinetics↗
PubMed1987

Intensity and anisotropy decays of the Wye base of yeast tRNA(Phe) as measured by frequency-domain fluorometry.

The intensity and anisotropy decays of Wye base fluorescence from yeast tRNA(Phe) were determined by frequency-domain fluorometry. The intensity decay is at least a double exponential in the presence and absence of Mg2+, but the multi-exponential character of the decay is more pronounced in the absence of Mg2+. The anisotropy decay displays components due to overall tRNA rotational diffusion and to local torsional motions. The amplitude of the local motion is decreased 2-fold by the presence of Mg2+. The results are broadly consistent with a more homogeneous environment for the Wye base in the presence of Mg2+.

Chelating Agents↗
PubMed1987

Demonstration of an associated anisotropy decay by frequency-domain fluorometry.

We used frequency-domain fluorometry to demonstrate the presence of an associated decay of fluorescence anisotropy. In such systems the individual correlation times are associated with distinct emitting species, each with its own characteristic lifetime and rotational correlation times. We obtained an associated system using 1-anilino-8-naphthalenesulfonic acid (ANS) in the presence of increasing amounts of apomyoglobin. When both free and apomyoglobin-bound ANS contributed to the emission the differential polarized phase angles become negative at particular frequencies, even though the fundamental anisotropy (r0) is greater than zero. Additionally, the modulated anisotropy decreases at high frequencies. Both observations appear to be the unique consequence of an associated anisotropy decay, and are not possible for a multiexponential anisotropy decay of a single species.

Anilino Naphthalenesulfonates↗
PubMed1988

Lifetime distributions and anisotropy decays of indole fluorescence in cyclohexane/ethanol mixtures by frequency-domain fluorometry.

We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.

Cyclohexanes↗
PubMed1990

Influence of end-to-end diffusion on intramolecular energy transfer as observed by frequency-domain fluorometry.

We investigated the influence of end-to-end diffusion on intramolecular energy transfer between a naphthalene donor and dansyl acceptor linked by polymethylene chain. A range of viscosities from 0.6 to 200 cP were obtained using propylene glycol at different temperatures (0-80 degrees C) and methanol at 20 degrees C. The intensity decays of naphthalene were measured in the frequency domain. Several theoretical models, including distance distributions, were used to fit the data. The results indicate that end-to-end diffusion of flexible donor-acceptor pairs can be detected and quantified using frequency-domain fluorometry, even in the presence of a distribution of donor-to-acceptor distances.

Diffusion↗
PubMed1991

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry.

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.

Mathematics↗
PubMed1991

Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry.

The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.

Cysteine↗
PubMed1992

Fluorescence lifetime distribution of 1,8-anilinonaphthalenesulfonate (ANS) in reversed micelles detected by frequency domain fluorometry.

The fluorescence emission decay of ANS (1,8-anilinonaphthalenesulfonate) in reversed AOT (sodium bis-(2-ethyl-1-hexy)sulfosuccinate) micelles at different water contents was investigated by frequency domain fluorometry. The whole ANS emission decay in reversed AOT micelles could not be fitted in terms of discrete lifetime values, i.e., mono-exponential and bi-exponential models. Better fits were obtained when using continuous unimodal Lorentzian lifetime distributions. This was interpreted as arising from the reorientation processes of water molecules around the excited state of ANS or probe exchange among different probe locations, occurring on a time scale longer than fluorophore lifetime. The dependence of ANS fluorescence anisotropy on the emission wavelength was consistent with the existence of a great emission heterogeneity especially for inverted micelles having reduced H2O/AOT molar ratio. Finally, the observation that the distribution width decreases with increasing temperature and/or micelle size suggested that fast processes of water dipolar reorganization around the fluorophore are facilitated under these conditions.

Anilino Naphthalenesulfonates↗