Steady-state fluorescence anisotropy

The rotational mobility of molecules in the excited state will induce the depolarization of light, causing excitation polarized light and fluorescence emission to have different angular displacements. 

The fluorescence anisotropy is the fraction of polarized light emitted by a fluorophore when excited with polarized light, as calculated by Equations (12.3) and (12.4)  

Fluorescence anisotropy can also be calculated by the quotient between the fluorescence intensities obtained at the four different combinations of the excitation and emission polarizer positions, as shown by Equation (12.3), Iw and Ihh account for the fluorescence intensity obtained with both polarizers at the vertical and horizontal position, respectively. Ihv corresponds to the fluorescence emission obtained with excitation polarizer horizontally and emission polarizer vertically oriented. In contrast, lyn corresponds to the fluorescence emission obtained with excitation polarizer verticahy and emission polarizer horizontally oriented. The G factor (Equation 12.4) is a parameter that accounts for the sensitivity of the detection system for verticahy and horizontally polarized lights [3]. 

Fluorescence anisotropy can be correlated with the rotational freedom of the fluorophores present in the sample. High anisotropy values correspond to a low depolarization of the fluorescence emitted light and are indicative of the presence of fluorophores with low rotational freedom. In contrast, low anisotropy values are a consequence of a high depolarization of the emitted light and are commonly ascribed to the presence of highly mobile fluorophores. 

When a fluorophore is inserted within a matrix (fluorophores in solid supports or bound to molecular chains, such as polymers or proteins) the fluorescence anisotropy is composed by different rotational contributions the self rotation of the fluorophore within the matrix and the overall rotation of the complex formed 

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Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

The physical dimensions and dynamics of calmodulin have also been investigated by tyrosine fluorescence. To learn about the internal mobility of calmodulin, Lambooy et al 1 and Steiner et al measured the steady-state fluorescence anisotropy of the tyrosine. Since the average correlation... 

H. Pottel, W. van der Meer, and W. Herreman, Correlation between the order parameter and the steady-state fluorescence anisotropy of l,6-diphenyl-l,3,5-hexatriene and an evaluation of membrane fluidity, Biochim. Biophys. Acta 730, 181-186 (1983). 

The modulation of synaptosomal plasma membranes (SPMs) by adriamycin and the resultant effects on the activity of membrane-bound enzymes have been reported [58]. Again DPH was used as fluorescence probe. Adriamycin increased the lipid fluidity of the membrane labeled with DPH, as indicated by the steady-state fluorescence anisotropy. The lipid-phase separation of the membrane at 23.3 °C was perturbed by adriamycin so that the transition temperature was reduced to 16.2 °C. At the same time it was found that the Na+,K+-stimulated ATPase activity exhibits a break point at 22.8 °C in control SPMs. This was reduced to 15.8 °C in adriamydn-treated SPMs. It was proposed that adriamycin achieves this effect through asymmetric perturbation of the lipid membrane structure and that this change in the membrane fluidity may be an early key event in adriamycin-induced neurotoxicity. 

Suzuki, H., Obara, M., Kubo, K., Kanazawa, T. (1989). Changes in the steady-state fluorescence anisotropy of N-iodoacetyl-N -(5-sulfo-l-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle. J. Biol. Chem. 264, 920-927. 

Figure 11.4 Steady-state fluorescence anisotropy vs. temperature/viscosity ratio for tryptophan residues of cytochrome b2 core. Data are obtained by thermal variations in the range 10-36°C.

FP is an alternative readout principle for endopeptidase activity assays. FP or anisotropy measurements allow the detection of changes in the rotational correlation time of particles. These differences in the rotational correlation (or relaxation) time are related to different masses of particles. The experimental determination of steady-state fluorescence anisotropy requires the linear polarization of the light used for the excitation of the probe as well as linear polarization of the emitted fluorescence. Based on data of an appropriate experiment, the fluorescence anisotropy can be calculated as ... 

Fig. 7.12. Steady-state fluorescence anisotropy as a function of the Zn +/monomer ratio. (Reprinted with permission from ref. [28]. Copyright 2005 American Chemical Society).

Fig. 17. Pressure dependence of the steady-state fluorescence anisotropy r g of TMA-DPH in DPPC/cholesterol unilamellar vesicles at different sterol concentrations (7 = 50 °C).

The fluorescent spectroscopy techniques were used to observe the interaction of dyes with macromolecules.5 The fluorescent spectra and steady-state fluorescence anisotropy of dyes in the presence of enzymes were determined with an Aminco-Bowman Series 2 spectrofluorimeter (ThermoSpectronic, USA) equipped with polarizers. 

Fig. 1. Steady-state fluorescent anisotropy r of xanthene (A) and anthracene (B) dyes in the presence of apo-obelin 1 - FS, 2 - EY, 3- EB (plotted to the right ordinate), 4 - CA, 5 - BA, 6 - IA (plotted to the right ordinate).

The steady-state fluorescence anisotropy/polarization method is also simple and relies on the fact that the probe molecule will tumble rapidly in solution when free, but will have restricted motion upon binding to a macromolecule. Optical excitation of the probe by polarized light will result in preferential absorption by those molecules whose absorption transition dipole is parallel to the electric field vector direction of the light. The subsequent fluorescence will be partially polarized. The definitions of anisotropy (r) and polarization (P) are [188]... 

Steady-state fluorescence anisotropy of Trp residues in both preparations of ai-acid glycoprotein was performed at different temperatures. The Perrin plots (Fig. 5.7 and 5.8) reveal that Trp residues of ai-acid glycoproteiii s display free motions while those of a 1-acid glycoprotein follow the global rotation of the protein. 

Steady-state fluorescence anisotropy of fluorophore residues, measured as a flmction of temperature (5 to 35°C) (Perrin plot), will give us information concerning the motion of the fluorophore within the protein (Weber, 1952). Wlien the fluorophore is tightly bound to the protein, its motion will correspond to that of the protein. In this case, the rotational correlation time will be equal to that of the protein. When the fluorophore exhibits significant motions when bound to the protein, the rotational correlation time determined from the Perrin plot will be lower than that of the protein and will be the results of two motions, that of the protein and of the segmental motion of the fluorophore. 

Steady-state fluorescence anisotropy of 10 pM of Calcofluor in the presence of 5 pM of ai -acid glycoprotein = 435 nm and Xqx 300 nm) was performed at different temperatures. A Perrin plot representation (Fig. 8.21a.) yields a rotational correlation time equal to 7.5 ns at 20 °C. This value is lower than that (16 ns) expected for a i-acid glycoprotein and thus indicates that calcofluor displays segmental motions independent of the global rotation of the protein. Thus, two motions contribute to the depolarization process, the local motion of the carbohydrate residues and the global rotation of the protein, i.e., a fraction of the total depolarization is lost due to the segmental motion, and the remaining polarization decays as a result of the rotational diffusion of the protein. 

Figure 8.21. Steady-state fluorescence anisotropy vs. temperature over viscosity for 5 pM Calcofluor in the presence of 10 pM sialylated ai-acid glycoprotein (A ex, 300 nm Xem, 435 nm) (plot a), and for 8.5 pM Calcofluor in the presence of 5.5 pM asialylated Uj- acid glycoprotein ((A ex, 300 nm, A em, 445 nm (plot b). The data shown are the mean values of two measurements, and they are obtained by thermal variation in the range 15-35°C. The ratio T/p is expressed in Kelvins/centipoise. Source Albani, J. R., Sillen A., Coddeville, B., Plancke, Y. D., and Engelborghs, Y. 1999, Carbohydr. Res. 322, 87-94.

Steady-state fluorescence anisotropy In low-viscosity solvents the rotational depolarization of low molecular weight compounds occurs on the picosecond timescale [124]. Since in this case the rotation is much faster than the fluorescence, the steady-state emission is unpolarised. If the rotational motion of the fluorophore is on the same timescale as the fluorescence, a steady state polarisation is observed. In the simplest case for a spherical rotor and a single-exponential fluorescence intensity decay (r), the measured anisotropy is given by... 

Fig. 6.27 Illustration of a Perrin plot for the determination of the apparent hydrodynamic volume V by steady-state fluorescence anisotropy measurements.

In the preceding ch t we described the measurement and interpretation of steady state fluorescence anisotropies. These values are measured using continuous illumination and r resent an average of the anisotropy decay ov - the intensity decay. Measurement of steady-state anisotropies is simple, but interpretation of the steady-state anisotropies usually d nds on an assumed form for the anisotropy decay, which is not directly observed in the experiment. Additional information is available if one measures the time-dependent anisotropy> that is, the values of r(t) following pulsed excitation. The form of the anisotropy decay depends on the size, shape, and flexibility of the labeled molecule, and the data can be compared with the decays calculated from various molecular models. Anisotropy decays can be obtained using the TD or the FD method. 

Figure 12.4 Schematic representation of the configuration of excitation and emission components during acquisition of steady-state fluorescence anisotropy, comprising light source, detector, excitation and emission monochromators (M xc, nd polarizers...

Measurements of steady-state fluorescence anisotropy were also performed for feed, retentate and permeate solutions of P-lactoglobulin obtained with both membranes (lOkDa, 30kDa) at different TMP values. The analysis of the fluorescence anisotropy data (Figure 12.13) shows, in comparison with the fluorescence anisotropy obtained for the feed solutions, a decrease in fluorescence anisotropy observed at all applied TMP values. 

Figure 12.13 Variation of the normalized steady-state fluorescence anisotropy, rM, acquired at /l,exc = 290nm and Aem = 350nrn for 3-lactoglobulin solutions, obtained after ultrafiltration with RC membranes of lOkDa (O) and SOkDa ( ), at different TMP values, with the respective retentates (A,A), in respect to feed ( ).

Figure 12.15 (a) Variation in the steady-state fluorescence anisotropy, r, acquired at Aexc = 290nm and Aem = 350nm for horseradish peroxidase (HRP-4C) solutions obtained after ultrafiltration with PES (O) and RC ( ) membranes, both with a cut-off of 30 kDa, at different TMP values, with the... 

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