Time-Resolved Anisotropy Measurements

It should be noted that the calculated anisotropy may not be applied to fs time-resolved anisotropy measurements because fs time-resolved experiments involve pumping and probing conditions and may involve overlapping between the vibronic structures of several electronic states due to the use of fs laser pulses. Nevertheless, we think the calculated anisotropy using Eq. (2.54) can provide a reference in comparing models. 

The results of fluorescence polarization studies of proteins were discussed above. Time-resolved anisotropy measurements often permit, without any additional variation of experimental conditions, intramolecular rotations to be distinguished from rotation of the whole protein molecule and characterized,... 

In time-resolved anisotropy measurements, the static or orientational components of motion and the rate of motion are derived. The time-resolved derivation of rs is revealed as... 

The rx term is the anisotropy at times long compared to the fluorescence lifetime, whereas in Eq. (5.9) 2 will be long. If there is no rM, then Eq. (5.8) reduces to the familiar Perrin equation for an isotropic rotator. Earlier, some confusion existed in this field since it was not recognized that an rro term was required for the case of membrane lipid bilayers. For the most part, time-resolved anisotropy measurements have a short rotational correlation time and an term. However, it has been recognized that a more adequate description may be to use two rotational correlation times, where the second may be quite long but not infinite as the rm implies/35 36 ... 

To get more insight into the effect of confinement on the binding between HPMO and the host, time-resolved anisotropy measurements have been carried out [58]. The result (Fig. 7.12) shows a remarkable difference in the anisotropy decays, especially for the HSA protein case. While in dioxane, the rotational time constant (45 ps) is close to the expected one using hydrodynamic theory [58], this time increases with the rigidity of the host (97 ps for a micelle, 154 ps for yS-CD... 

Fluorescence anisotropy measurements can also be used to obtain the rates of the excited state tautomerization. Two variants can be applied. The first is based on the analysis of time-resolved anisotropy curves. These are constructed from measurements of the fluorescence decay recorded with different positions of the polarizers in the excitation and emission channels. The anisotropy decay reflects the movement of the transition moment and thus, the hydrogen exchange. For molecules with a long-lived Sj state, the anisotropy decay can also be caused by rotational diffusion. In order to avoid depolarization effects due to molecular rotation, the experiments should be carried out in rigid media, such as polymers or glasses. When the Sj lifetime is short compared to that of rotational diffusion, tautomerization rates can be determined even in solution. This is the case for lb, for which time-resolved anisotropy measurements have been performed at 293 K, using a... 

Time-resolved anisotropy measurements of excitation hopping between two anthryl moieties attached to both ends of alkane molecules have been interpreted by a model based on conformational... 

Time-Resolved Anisotropy Measurements Time-resolved anisotropy measurements (TRAMs) offer a distinct advantage over their steady-state... 

First, we studied the solvent relaxation in solutions of diblock copolymer micelles. A commercially available polarity-sensitive probe, patman (Fig. 10, structure I), frequently used in phospolipid bilayer studies [123], was added to aqueous solutions of PS-PEO micelles. The probe binds strongly to micelles because its hydrophobic aliphatic chain has a strong affinity to the nonpolar PS core. The positively charged fluorescent headgroup is supposed to be located in the PEO shell close to the core-shell interface. The assumed localization has been supported by time-resolved anisotropy measurements. 

The time-resolved anisotropy measurements performed for pH 2 and 4 revealed fairly high values of the residual anisotropy (0.2 and 0.3, respectively), which indicates high rigidity of the microenvironment. 

In this chapter we describe the fundamental theory for steady-state measurements of fluorescence anisotropy and present selected biochemical applications. In the next chapter we will describe the theory and applications of time-resolved anisotropy measurements. 

In the preceding two chapter we described steady>state and time>resolved anisotropy measurements and presented a number of biochemical examples which illustrated the types of information available from these measurements. Throughout these chapters, we stated that anisotropy decay deprads on the size and shape of the rotating species. However, the theory which relates the form of the anisotropy decay to the shape of the molecule is complex and was not described in detail. In the present chapter we provide an overview of the rotational properties of non-spherical molecules, as well as representative examples. 

The confinement and microviscosity of the IL pool in IL/O microemulsions can have profound effects on its molecnlar dynamics. Time-resolved anisotropy measurements provide useful information on dynamics by monitoring the rotational relaxation of an excited dye molecule, such as coumarin-based fluorophores. Time-resolved fluorescence anisotropy can be calcnlated using Equation 19.1 ... 

The correlation time parameter, is especially relevant in time-resolved anisotropy measurements, when the discrimination of the individual rotation ability of the diflerent chemical species is possible. 

For fluorescence measurements, by far the most versatile and widely used time-resolved emission technique involves time-correlated single-photon counting [8] in conjunction with mode-locked lasers, a typical mo m apparatus being shown in Figure 15.8. The instrument response time of such an apparatus with microchannel plate detectors is of the order of 70 ps, giving an ultimate capability of measurement of decay times in the region of 7 ps. However, it is the phenomenal sensitivity and accuracy which are the main attractive features of the technique, which is widely used for time-resolved fluorescence decay, time-resolved emission spectra, and time-resolved anisotropy measurements. Below ate described three applkations of such time-resolved measurements on synthetic polymers, derived from recent work by the author s group. 

Fast librational motions of the fluorophore within the solvation shell should also be consideredd). The estimated characteristic time for perylene in paraffin is about 1 ps, which is not detectable by time-resolved anisotropy decay measurement. An apparent value of the emission anisotropy is thus measured, which is smaller than in the absence of libration. Such an explanation is consistent with the fact that fluorescein bound to a large molecule (e.g. polyacrylamide or monoglucoronide) exhibits a larger limiting anisotropy than free fluorescein in aqueous glycerolic solutions. However, the absorption and fluorescence spectra are different for free and bound fluorescein the question then arises as to whether r0 could be an intrinsic property of the fluorophore. 

In principle, the shape parameters of asymmetric rotors can be estimated from time-resolved anisotropy decay measurements, but in practice it is difficult to obtain accurate anisotropy decay curves over much more than one decade, which is often insufficient to determine more than two rotational correlation times. 

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. 

Anisotropy measurements yield information on molecular motions taking place during the fluorescence lifetime. Thus, measuring the time-dependent decay of fluorescence anisotropy provides information regarding rotational and diffusive motions of macromolecules (Wahl and Weber, 1967). Time-resolved anisotropy is determined by placing polarizers in the excitation and emission channels, and measuring the fluorescence decay of the parallel and perpendicular components of the emission. 

Thus, as mentioned earlier, time-resolved depolarization measurements afford a means of recording the time profile of the rotational autocorrelation function. The steady state technique, with continuous sample excitation, produces merely the time average of the emission anisotropy, F. For a rotating chromc hore with a sin e fluorescence decay time Tf, F is related to r(t) by the following expression... 

A further application of time-resolved fluorescence measurements is in the study of conformational dynamics of polymer chains in solution. Fluorescence anisotropy measurements of macromolecules incorporating suitable fluorescent probes can give details of chain mobility and polymer conformation (2,14). A particular example studied in this laboratory is the conformational changes which occur in aqueous solutions of polyelectrolytes as the solution pH is varied (15,16). Poly(methacrylic acid) (PMA) is known to exist in a compact hypercoiled conformation at low pH but undergoes a transition to a more extended conformation at a degree of neutralization (a) of 0.2 to 0.3 (1 6). Similar conformational transitions are known to occur in biopolymer systems and consequently there is considerable interest in understanding the nature of the structures present in model synthetic polyelectrolyte solutions. 

D.J.S. Birch, A.S. Holmes, J.R. Gilchrist, R.E. Imhof, S.M. Al-Alawi, B. Nadolski, A multiplexed single-photon instrument for routine measurement of time-resolved anisotropy, J. Phys. E, Sci. Instram. 20, 471 73 (1987)... 

E. Deprez, P. Tauc, H. Leh, J-F. Mouscadet, C. Auclair, J-C. Brochon, Oligomeric states of the HIV-1 integrase as measured by time-resolved anisotropy, Biochemistry 39, 9275-9284 (2000)... 

At present, there is widespread interest in directly measuring the time-dependent processes. This is because considerably more information is av lilable from the time-dependent data. For example, the time-dependent decays of protein fluorescence can occasionally be used to recover the emission spectra of individual tryptophan residues in a protein. The time-resolved anisotropies can reveal the shape of the protein and/or the extent of residue mobility within the protein. The time-resolved energy transfer can reveal not only the distance between the donor and acceptor, but also the distribution of these distances. The acquisition of such detailed information requires high resolution instrumentation and careful data acquisition and analysis. 

In the time-domain the anisotropy decay is obtained from the time-resolved decays of the parallel and perpendicular polarized components of the emission. More specifically, one measures the time-resolved decays of the parallel ( ) and the perpendicular ( ) components of the emission, and calculates the time-resolved anisotropy. 

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