Intrinsic fluorescence measurements

Rush et al.51 first described the effect of thermally induced conformational changes on migration behavior of a-lactalbumin. A sigmoidal dependency of the viscosity-corrected mobility on temperature was observed. Transition temperature also agreed closely with that determined by intrinsic fluorescence measurements. 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Schaferling M, Duerkop A (2008) Intrinsically referenced fluorimetric sensing and detection schemes methods, advantages and applications. In Resch-Genger U (ed) Standardization and quality assurance in fluorescence measurements I Springer Ser Fluoresc 5 373 -14... 

We can just measure the intrinsic fluorescence of the target analyte or design sensors based on the variation of the fluorescence of an indicator dye, with the determinand concentration. In the latter case, the probe molecule... 

L. Zheng and J.D. Brennan, Measurement of intrinsic fluorescence to probe the conformational flexibility and thermodynamic stability of a single tryptophan protein entrapped in a sol-gel derived glass matrix. Analyst 123, 1735-1744 (1998). 

Temporal characteristics at early stages were elucidated by measuring fluorescence intensity with the gate time of 1.74 ns as a function of the delay time. Compared to the laser pulse, the time where the maximum intensity is attained shifts to the early stage as the laser fluence becomes high. Of course, we could not find out any decay component with intrinsic fluorescence lifetime of 17 and 35 ns. It is concluded that an Si - Si annihilation occurs quite efficiently during the pulse width. 

In the passive mode, the optical device measures the variation in fluorescence characteristics (intensity, lifetime, polarization) of an intrinsically fluorescent analyte. The optical device can have different optical configurations involving in most cases an optical fiber (passive optode) (Figure 10.44). 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

FLUORESCENCE MEASUREMENTS OF LIGAND BINDING. In principle, ligand binding may either enhance or quench the intrinsic or extrinsic fluorescence of its macromolecular receptor or it may change the polarization of the fluorescence emission (see below). 

Figure 4.5 — Generic manifold for implementation of methods using fluorimetric sensors for directly measuring the intrinsic fluorescence of the analyte (singlechannel system) or a reaction product (by including the zone bound by the dotted line for reagent supply).

The fluorescence of liquid alkanes is supposed to originate entirely from the relaxed Si state. Walter and Lipsky [154], by measuring the fluorescence yields of alkane solutions irradiated with 165 nm photons or Kr beta particles ( niax = 0-67 MeV) relative to benzene fluorescence, determined the following yields 2.3-dimethylbutane G Si) < 1.3, cyclohexane 1.4-1.7, methylcyclohexane 1.9-2.2, dodecane 3.3-3.9, hexadecane 3.3-3.9, d5-decalin 3.4, and bicyclohexyl 3.5. After reinvestigating the intrinsic quantum yield of cyclohexane fluorescence, Choi et al. published G(5 i) = 1.45 for this alkane in Ref. 155. For tra 5-decalin a G Si) value of 2.8-3.1 has been accepted [65,128,132]. The uncertainties in the values reflect the uncertainties in the intrinsic fluorescence quantum yields. 

Protein concentration can also be determined by measuring the intrinsic fluorescence based on fluorescence emission by the aromatic amino acids tryptophan, tyrosine, and/or phenylalanine. Usually tryptophan fluorescence is measured. The fluorescence intensity of the protein sample solution is measured and the concentration is calculated from a calibration curve based on the fluorescence emission of standard solutions prepared from the purified protein. This assay can be used to quantitate protein solutions with concentrations of 5 to 50 (J-g/ml. 

Measurement of intrinsic fluorescence by aromatic amino acids is primarily used to obtain qualitative information (Freifelder, 1982). However, with a protein standard whose aromatic amino acid content is similar to that of... 

When the absorptivity for a protein is known, the A28o and 4205 measurements require <30 min depending on the number of samples. When standards are used for quantitation with these assays or for intrinsic fluorescence quantitation, 1 hr is required. 

Phillips, W. J., and Cerione, R. A. (1988). The intrinsic fluorescence of the a subunit of transducin. Measurement of receptor-dependent guanine nucleotide exchange./ Biol. Chem. 263, 15498-15505. 

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