Fluorescence polarization effects

Figure 4. Continuous analysis by fluorescence polarization of FLPEP binding and dissociation to permeabilized neutrophils. Each curve represents the average of two determinations. The descending curves represent the effect of the addition of receptor antagonist after FLPEP had bound to the receptors. Upper panel, in the absence of GTPyS, receptor antagonist added at t = 60 s lower panel, in the presence of GTPyS, antagonist added at t =...

Growth of the degree of fluorescence polarization (the Weber s effect) and a decrease of energy transfer efficiency while shifting the excitation wavelength to the red edge. 

Papahadjopoulos, D., Jacobson, K., Nir, S. and Isac, T. (1973). Phase transitions in phospholipid vesicles fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol, Biophys. Biochim. Acta, 311, 330-348. 

We have considered spherical molecules so far, but it should be noted that isotropic rotations can also be observed in the case of molecules with cylindrical symmetry and whose absorption and emission transition moments are parallel and oriented along the symmetry axis. In fact, any rotation around this axis has no effect on the fluorescence polarization. Only rotations perpendicular to this axis have an effect. A typical example is diphenylhexatriene whose transition moment is very close to the molecular axis (see Chapter 8). 

Attention should be paid to possible problems in the measurement of fluorescence quantum yields (some of which are discussed Section 6.1.5) inner filter effects, possible wavelength effects on Op, refractive index corrections, polarization effects, temperature effects, impurity effects, photochemical instability and Raman scattering. 

Pitfalls in steady-state fluorescence measurements inner filter effects and polarization effects... 

Polarization effects The transmission efficiency of a monochromator depends on the polarization of light. This can easily be demonstrated by placing a polarizer between the sample and the emission monochromator it is observed that the position and shape of the fluorescence spectrum may significantly depend on the orientation of the polarizer. Consequently, the observed fluorescence intensity depends on the polarization of the emitted fluorescence, i.e. on the relative contribution of the vertically and horizontally polarized components. This problem can be circumvented in the following way. 

Appendix Elimination of polarization effects in the measurement of fluorescence intensity and lifetime... 

In conclusion, the method of intramolecular excimer formation is rapid and convenient, but the above discussion has shown that great care is needed for a reliable interpretation of the experimental results. In some cases it has been demonstrated that the results in terms of equivalent microviscosity are consistent with those obtained by the fluorescence polarization method (described in Section 8.5), but this is not a general rule. Nevertheless, the relative changes in fluidity and local dynamics upon an external perturbation are less dependent on the probe, and useful applications to the study of temperature or pressure effects have been reported. 

Figure 6.1. Fluorescence polarization immunoassay for theophylline. (A) Effect of theophylline rabbit antiserum ( ) and normal rabbit serum (A) on the fluorescence polarization of the theophylline-umbelliferone conjugate. (B) Fluorescence polarization of the theophylline-umbelliferone conjugate in the presence of varying concentrations of theophylline. (Reprinted from Ref. 1, with permission from Academic Press.)...

Acetone, methyl ethyl ketone (2-butanone) and methyl isobutyl ketone (4-methyl-pentan-2-one) (6.8 mmol/kg bw for 3 days) increased the hepatotoxicity of carbon tetrachloride to Sprague-Dawley rats (Raymond Plaa, 1995a) this enhancement of toxicity was coincident with increased microsomal aniline hydroxylase activity (Raymond Plaa, 1995b). In addition to the effect on cytochrome P450, acetone, but not the other ketones, increased basal canalicular membrane fluidity, as measured by fluorescence polarization of 1,6-diphenyl-1,3,5 -hexatriene or 1 - [4-(trimethylammoniumphenyl)-6-phenyl] -1,3,5 -hexa-triene (Raymond Plaa, 1996). 

Sample preparation was given elsewhere [2]. Femtosecond fluorescence upconversion and picosecond time-correlated single-photon-counting set-ups were employed for the measurement of the fluorescence transients. The system response (FWHM) of the femtosecond fluorescence up-conversion and time-correlated single-photon-counting setups are 280 fs and 16 ps, respectively [3] The measured transients were fitted to multiexponential functions convoluted with the system response function. After deconvolution the time resolution was 100 fs. In the upconversion experiments, excitation was at 350 nm, the transients were measured from 420 nm upto 680 nm. Experiments were performed under magic angle conditions (to remove the fluorescence intensity effects of rotational motions of the probed molecules), as well as under polarization conditions in order to obtain the time evolution of the fluorescence anisotropy. 

The solvent pH and polarity will affect the absorbance and fluorescence properties of a protein. A notable example of pH effects on absorbance is seen with tyrosine residues, where a change in pH from neutral to alkaline results in a shift of the absorbance maximum to a longer wavelength and an increase in absorptivity due to dissociation of the tyrosine phenolic hydroxyl group (Freifelder, 1982 Fasman, 1989). An example of solvent polarity effects on fluorescence is observed with tryptophan, where a decrease in solvent polarity... 

The medium in which a species is dissolved or on which it is adsorbed may exert considerable influence on the intensity and wavelength of the fluorescence. Polar materials such as alcohols or esters frequently increase the intensity of the fluorescence relative to non-polar hydrocarbon solvents. The solvent environment often prevents or inhibits intersystem crossing to a triplet state in favour of excitation to a singlet state and fluorescence, while in many cases the opposite is true. The dielectric constant of solvents has been shown to influence the fluorescence intensity and wavelength maxima of some compounds [33,34]. Fig.2.9 shows the effect of solvent dielectric constant on the fluorescence intensity of DNS-phenol, while Table 2.4 shows the corresponding effect on the fluorescence wavelength [34]. For DNS-phenol, solvents of low dielectric constants result in the most intense fluorescence and shift the wavelength maxima to lower values. 

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