Steady-state polarization anisotropy measurements

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] ... 

Measurement of steady-state emission anisotropy. Polarization spectra... 

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 ... 

The measurement of steady-state anisotropy r is simple and needs two polarizers, one in excitation and the other in emission beams. When the sample is excited... 

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

At the present time, two methods are in common use for the determination of time-resolved anisotropy parameters—the single-photon counting or pulse method 55-56 and the frequency-domain or phase fluorometric methods. 57 59) These are described elsewhere in this series. Recently, both of these techniques have undergone considerable development, and there are a number of commercially available instruments which include analysis software. The question of which technique would be better for the study of membranes is therefore difficult to answer. Certainly, however, the multifrequency phase instruments are now fully comparable with the time-domain instruments, a situation which was not the case only a few years ago. Time-resolved measurements are generally rather more difficult to perform and may take considerably longer than the steady-state anisotropy measurements, and this should be borne in mind when samples are unstable or if information of kinetics is required. It is therefore important to evaluate the need to take such measurements in studies of membranes. Steady-state instruments are of course much less expensive, and considerable information can be extracted, although polarization optics are not usually supplied as standard. 

Steady-state fluorescence polarization studies have been carried out with a number of peptides, including model peptides, ACTH, glucagon, melittin, and thyrocalcitonin. This work has been reviewed 5 and will not be discussed in the present article. More recently, interesting information on the rotational behavior and structural flexibility of various peptides has been obtained from fluorescence anisotropy decay measurements. 

For quite some time, there have been indications for a phase-separation in the shell of polyelectrolyte block copolymer micelles. Electrophoretic mobility measurements on PS-PMAc [50] indicated that a part of the shell exhibits a considerable higher ionic strength than the surrounding medium. This had been corroborated by fluorescence studies on PS-PMAc [51-53] and PS-P2VP-heteroarm star polymers [54]. According to the steady-state fluorescence and anisotropy decays of fluorophores attached to the ends of the PMAc-blocks, a certain fraction of the fluorophores (probably those on the blocks that were folded back to the core/shell interface) monitored a lower polarity of the environment. Their mobility was substantially restricted. It thus seemed as if the polyelectrolyte corona was phase separated into a dense interior part and a dilute outer part. Further experimental evidence for the existence of a dense interior corona domain has been found in an NMR/SANS-study on poly(methylmethacrylate-fr-acrylic acid) (PMMA-PAAc) micelles [55]. 

Steady-State Anisotropy Following continuous excitation with vertically polarized light, a distribution of fluorophores whose transition vectors for the absorption process are vertically aligned will be photoselected, creating an excited state population, which possesses a degree of anisotropy (r) or optical order, in an otherwise isotropic distribution of fluorophores. Measurement of the intensity of fluorescence, via an emission polarizer in planes parallel (z n) and perpendicular (zx) to the vertical plane allows estimation of r from... 

Figure 5.7. Steady-state fluorescence polarization versus temperature over viscosity ratio for Trp residues of human aj -acid glycoprotein prepared by acetonic precipitation. Data were obtained by thermal variations in the range 7-35" C. Xex = 300 nm. Xem = 330 nm. Protein concentration is equal to 10 pM. The rotational correlation time determined from the Perrin plot is equal to 13 ns at 20°C is in the same range as that (17 ns) expected for the protein at the same temperature, indicates the presence of residual motions. Also, the extrapolated anisotropy (0.264) is equal to that measured at -35 C (0.267). Source Albani, J. R. 1998, Spectrochimica Acta, Part A. 54, 173-183.

The appeal of fluorescence spectroscopy in the study of biomolecular systems lies in the characteristic time scale of the emission process, the sensitivity of the technique, and its ability to accommodate rapid and facile changes in the solvent milieu under conditions corresponding to thermodynamic equilibrium. The time scale of the emission process invites exploitation in two related manners. First, information on hydrodynamic aspects of the system is available from steady-state or time-resolved measurements. Second, detailed information on local dynamic processes within the biomolecular matrix may be derived. Information on hydrodynamic aspects of a macromolecular system may be used to study binding processes, that is, the association of small ligands with macromolecules or macromolecule-macromolecule interactions. In this chapter we focus on the latter applications of polarization or anisotropy data. We shall also try to clarify aspects of this area that our experience has shown to be occasionally misunderstood by initiates. 

Time resolved anisotropies can simply be measured by adding polarization sensitivity to time resolved fluorescence measurements described in Section 2.7.4. The qualitative interpretation of the data follows intuitively from the discussion of steady state anisotropy in Section 2.7.5, so we do not describe the method further. For more information we refer the reader elsewhere [1,127]. 

Fluorescence measurements. Steady-state fluorescence polarization measurements were performed with a SIi4 8000 spectrofluorimeter at 20 C. Vesicles were labelled with 1 yM DPH by incubation for 30 min in the dark, at room temperature. Data were expressed as plots of the anisotropy parameter [(ro/r - which provides a quantitative index of the environmental... 

Hence, it is possible to construct a standard curve relating viscometric measurements to steady-state anisotropy measurements for a particular fluid and use the quantitative relationship to determine the viscosity of the fluid by fluorescence polarization [4,9-11]. A standard curve in a reference calibration oil such as white paraffin oil can be used to determine the viscosity of another fluid as long as the calibration fluid is similar in dielectric constant and viscosity to the fluid being analyzed. It is important to keep in mind that the same fluorescent probe may display different behavior even in different hydrocarbon calibration oils, hence one must exercise caution when determining absolute values for microvis-... 

This ratio is an indication of the structural order of the fluid matrix, and for an isotropic fluid it should be close to unity [4]. In order to differentiate between anisotropy of the medium and that of the molecular rotations, one should compare the V vs. To plots (determined from the steady-state anisotropy measurements at low temperatures in high density fluids at different excitation wavelengths) for a particular fluorophore embedded in an isotropic medium to a similar plot for the probe embedded in the test fluid. If, for example, a strong dependence of v on the value of ro is evident for both media and the ratio of in-plane to out-of-plane rotational rates is very high (>10), one can conclude that the rotations are anisotropic but the medium is isotropic. The technique of differential polarized phase fluorometry [8,12], which is beyond the scope of this chapter, has been successfully applied to study the types of rotations displayed by fluorophores embedded in different media. 

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