Time-resolved anisotropy decay

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. 

The limiting values of the fluorescence anisotropy are 0.4 and -0.2, corresponding to parallel and perpendicular absorption and emission dipoles, respectively. For a chromophore undergoing internal conversion, the change in angle between the two vectors can be instantaneous. It may also be caused by energy transfer or physical motion. On the laboratory scale, the time-resolved anisotropy decay is determined as ... 

When a fluorophore exhibits segmental motions, time-resolved anisotropy decay must be analyzed as the sum of exponential decays ... 

The Pauli Master equation approach to calculating RET rates is particularly useful for simulating time-resolved anisotropy decay that results from RET within aggregates of molecules. In that case the orientation of the aggregate in the laboratory frame is also randomly selected at each Monte Carlo iteration in order to account for the rotational averaging properly. 

For example, 2-aminopurine (AP) has been used to probe the dynamics of mismatches in DNA [340]. AP can be excited at 320 nm, where the normal DNA bases do not absorb (much), and emits at 380 nm (Fig. 4.36). Time-resolved anisotropy decays of AP across from all four natural DNA bases were performed (Fig. 4.37). AP can hydrogen-bond to T nearly as well as the natural A. The data were fitted to sums of two exponential terms the long time corresponded to overall tumbling and the short time to local motions within the DNA base stack. It was found that the internal correlation time corresponding to local motions, at 4°C,... 

Time resolved anisotropy decay performed at different temperatures, reveals the presence of two rotational correlation times, one Op, corresponding to the global rotation of the protein and the second Oa, a shorter one, reveals the presence of local residual motions around and / or near the two tryptophan residues. Oa is an apparent rotational time that is a mathematical combination of the global rotation of the protein and the segmental motion of the fluorophore. 

Time-resolved anisotropy decay may also be used to obtain information on the dynamics of Trp residues. However, the technique does not give detailed information on the motion of each class of Trp residues. When the fluorophore has segmental motions, the anisotropy will be analyzed as a sum of exponential decays ... 

Fig. 3. Effect of melittin binding to Ca -ATPase in sarcoplasmatic reticulum on the time-resolved anisotropy decay of the membrane probe diphenylhexatriene. (From Voss et al. )...

Fipipe 17.26. Time-resolved anisotropy decays of aimexin V in the Fi9ire17.28. Structure of aimexinV in the absence (top) and presence... 

In both cases, the time-resolved anisotropy decay acquires a complex form of an infinite series. Gordon [77] has shown that the decay may, in special cases, exhibit damped oscUlatiOTis, which has been observed experimentally, but the original RD model was unable to offer an explanation of this rather exotic behavior. Numerical calculations by McClung [79] validated the ED prediction for spherical molecules. For asymmetric molecules, the calculations confirmed the formulae based on the J-diffusion model and indicated that the assumptions used in the M-diffusion model are less realistic [81]. 

In principle, pulsed excitation measurements can provide direct observation of time-resolved polarization decays and permit the single-exponential or multiexponential nature of the decay curves to be measured. In practice, however, accurate quantification of a multiexponential curve often requires that the emission decay be measured down to low intensity values, where obtaining a satisfactory signal -to-noise ratio can be a time-consuming process. In addition, the accuracy of rotational rate measurements close to a nanosecond or less are severely limited by tbe pulse width of the flash lamps. As a result, pulsed-excitation polarization measurements are not commonly used for short rotational periods or for careful measurements of rotational anisotropy. 

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. 

The time-resolved anisotropy. Fig. 7.13, decays from an initial value of 0.291 to a constant value (0.042) that is reached in about 2 ns. The final value is exactly one-... 

Figure 7.7 Time-resolved hydration process for the proteins Sublitisin Carlsberg (SC) and Monellin (Mn). The time evolution of the constructed correlation function is shown for the protein SC (top), the Dansyl dye bonded SC (middle), and for the protein Mn (bottom). The inset of each part shows the corresponding time-resolved anisotropy r(t) decay [21].

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... 

However, time-resolved optical spectroscopy is perhaps the premier method for learning about the dynamics of a complex system, especially on nanosecond or picosecond time scales. Some DNA dynamics data from NMR spectroscopy are presented in Table 4.3. Time-resolved emission decays, time-resolved fluorescence anisotropy, and time-resolved Stokes shifts measurements of probe molecules in DNA have been described (and see below) and fast components in the time decays assigned to various DNA motions. The dynamics as a function of sequence are incompletely mapped and provide an exciting area for future investigations. 

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. 

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. 

Keywords Steady-state fluorescence spectra Time-resolved fluorescence decays Fluorescence anisotropy Huorescence quenching Nonradiative excitation energy transfer Solvent relaxation Excimer J and H aggregates... 

Soutar et al. [125] measured the time-resolved anisotropy, r(f), for dilute solutions of PNIPAM, randomly labeled by ca. 5 mol.% acenaphthylene in water, methanol, and in the whole region of their mixtures at temperatures covering the LCST dependence (see Fig. 7) on the composition of the solvent mixture. They found that the anisotropy decays are double exponential in all cases. They used the string of pearls model for the system in pure water and in water-methanol mixtures, where they ascribed the short time to the rotation of probes in the highly solvated parts of the PNIPAM chain and the long time to the rotation of probes in coiled dehydrated parts. Because they also observed the double-exponential... 

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