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At least 523 records · Page 29Linked to original sources
PubMed1983

Picosecond laser fluorometry of FAD of D-amino acid oxidase-benzoate complex.

Formation of a complex of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, an enzyme-substrate complex model, was studied by measuring the fluorescence life-time of the coenzyme FAD of the complex by using a mode-locked Nd:YAG laser and a streak camera. The value of lifetime was 60 +/- 10 ps in the monomer of the complex and it was extremely short (much less than 5 ps) in the dimer of the complex. Since the values of fluorescence lifetime of the coenzyme are 130 ps in the monomeric form of free enzyme and 40 ps in the dimeric form of free enzyme, the decrease in the lifetime upon complex formation with benzoate is slight in the monomer (reduced to one-half) whereas marked in the dimer (reduced to less than 1/10). By analyzing the fluorescence decay curve, a dissociation constant of the monomer-dimer equilibrium of the complex was evaluated to be 0.4 +/- 0.3 microM, which is much smaller than that in free enzyme. Fluorescence analysis under steady state excitation revealed that the apparent dissociation constant (K) of FAD from the enzyme was decreased by 1:1000 upon the complex formation. Relative quantum yield of the fluorescence of FAD in the complex to that of free FAD exhibited appreciable dependence on the complex concentration: greater in the monomer and less in the dimer. These results suggest that a molecular interaction between FAD and amino acid residue(s) is strengthened by the complex formation, which contributes to a remarkable conformational change in the protein moiety of the complex.

Benzoates↗
PubMed1984

A medicolegal study on enzymic fluorometry of choline in human semen.

Studies have been conducted on an enzymic fluorometric method based on an initial rate of reaction for the determination of choline. The reaction system consists of choline oxidase coupled to peroxidase and homovanillic acid. Concentrations of choline as low as 0.1 nmol could be detected by this procedure. The concentration of free choline in normal semen was 18.7 to 29.5 mumol/mL. Free choline in other body fluids was negligible. The choline concentrations in seminal stains maintained at room temperature were not changed during a 30-day period. Those concentrations in seminal fluids kept at room temperature were detected until at least the fifth day.

Alcohol Oxidoreductases↗
PubMed1984

Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry.

We report the first time-resolved fluorescence emission spectra and time-resolved fluorescence anisotropies obtained using frequency-domain fluorescence spectroscopy. We examined the fluorophore p-2-toluidinyl-6-naphthalenesulfonic acid (TNS) in viscous solvents and bound to the heme site of apomyoglobin using multifrequency phase fluorometers. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 1 to 200 MHz. For time-resolved emission spectra, the impulse response for the decay of intensity at each emission wavelength was obtained from the frequency response of the sample at the same emission wavelength. The decays have negative pre-exponential factors, consistent with a time-dependent spectral shift to longer wavelengths. These multiexponential decays were used to construct the time-resolved emission spectra, which were found to be in good agreement with earlier spectra obtained from time-domain measurements. Additionally, time-resolved anisotropies were obtained from the frequency-dependent phase angle differences between the parallel and perpendicularly polarized components of the emission. The rotational correlation times of TNS bound to apomyoglobin are consistent with those expected for this probe rigidly bound to the protein. TNS in propylene glycol also displayed a single exponential decay of anisotropy. These results, in conjunction with the previous successful resolution of multiexponential decays of fluorescence intensity (Lakowicz, J. R., Gratton, E., Laczko, G., Cherek, H., and Limkeman, M. (1984) Biophys. J., in press; Gratton, E., Lakowicz, J. R., Maliwal, B. P., Cherek, H., Laczko, G., and Limkeman, M. (1984) Biophys. J., in press) demonstrate that frequency-domain measurements provide information which is, at a minimum, equivalent to that obtainable from time-domain measurements.

Animals↗
PubMedDate not supplied

Specific determination of alpha-tocopherol in food and feed by fluorometry. Part 1. Manual method.

The proposed fluorometric method for determining alpha-tocopherol is highly specific and sensitive, yet requires low-cost equipment available in any laboratory. It is robust and fairly fast (4 determinations in 100 min, sample preparation not included). It has been tested in parallel with a conventional thin layer chromatographic method on foods and feeds. The only necessary cleanup is the usual saponification. The unsaponifiable fraction can be extracted with ethyl ether or, preferably, with Extrelut columns. Isooctane is used as a carrier solvent. Reagents and their solvents are added to the isooctane solution before each successive reaction and are then eliminated by partition with water. The alpha-tocopherol (alpha-T) derivative always remains in isooctane. The first step is nitrosation and elimination of tocopherols and tocotrienols other than alpha-isomers. alpha-T is then oxidized to alpha-tocored (alpha-TR) with a mixture of sulfuric acid, ferric chloride, and iodine bromide. alpha-TR is then condensed to a new reagent: 4,5-dimethyl-o-phenylenediamine. The phenazine formed is strongly fluorescent. Iodine and bromine add to the double bonds of alpha-tocotrienol present and quench the fluorescence of its phenazine. A procedure for blank assays specifically inhibits the conversion of alpha-T to alpha-TR.

Animal Feed↗
PubMed1978

[The microscopy fluorometry (author's transl)].

The microscope photometry and the method of continuously recording photometry help in the exemplification of the temporal development of virus multiplication and in providing information on the localization of the antigen in cells. Furthermore an automatic fluorometric microscope photometer (AFM) is described. By this method, the titre and that dilution required for the complete detection of antigens can be determined and an objective comparison of the various conjugate specimens is possible.

Antigens↗
PubMed1982

Fluorometry of selenium in serum or urine.

This fluorometric procedure for determining selenium in human serum or urine is sensitive (requiring only 0.4 mL of sample), accurate, simple, and can be performed on several samples concurrently. Using this technique, we found a mean selenium concentration in the serum of normal Canadian men of 142.9 (SD 16.1) micrograms/L. The mean urinary excretion rate was 124.5 (SD 76.0) micrograms/day.

Adult↗
PubMed1980

Fluorometry of ethylene glycol in serum.

We describe an assay for ethylene glycol in serum, which is based on the Hantzsch condensation reaction. Ethylene glycol is dehydrogenated by sodium periodate to form two molecules of formaldehyde, which is then combined with ammonium ion and acetylacetone to form a fluorescent diacetyllutidine derivative. The detection limit of this method is 50 mg/L. The procedure is sensitive, requires only 100 micro L of serum, and the standard curve is linear over a working range of 50 to 250 mg/L. Average analytical recovery ranged from 99 to 102% and within-run precision studies showed CVs of < 5%. Reagents are easily prepared and available.

Ethylene Glycols↗