Fluorescence depolarization rotational diffusion

The nmr data for this type of motion are direct and the motion clearly involves rotation about bonds in the millisecond time scale range. However. less direct evidence for motion comes from other techniques such as fluorescence depolarization, 02 diffusion, hydrogen exchange kinetics, and nmr relaxation times (see Ref. 4). The extent of this motion is not yet easy to define, but this evidence points to motion in the nanosecond time scale range. It is tempting to see the motion in this time scale as bond oscillations rather than rotations. To put it in a different way, on this time scale the side chains have some freedom to move with respect to each other but not normally to undergo substantial bond rotation. Table IV summarizes some references for motion of different types. Additionally, nmr relaxation studies suggest that the backbone or main chain of a protein is more restricted than that of the side chains. 

Photoexcited fluorescence from spread monolayers may be studied [158,159] if the substance has both a strong absorption band and a high emission yield as in the case for chlorophyll [159]. Gaines and co-workers [160] have reported on the emission from monolayers of Ru(bipyridine)3, one of the pyridine ligands having attached C g aliphatic chains. Ruorescence depolarization provides information about the restriction of rotational diffusion of molecules in a monolayer [161], Combining pressure-area... 

Tao T 1969 Time-dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolecules Biopolymers 8 609-32... 

One can employ linearly polarized light to excite selectively those fluorophores that are in a particular orientation. The difference between excitation and emitted light polarization changes whenever fluorophores rotate during the period of time between excitation and emission. The magnitude of depolarization can be measured, and one can therefore deduce the fluorophore s rotational relaxation kinetics. Extrinsic fluorescence probes are especially useful here, because the proper choice of their fluorescence lifetime will greatly improve the measurement of rotational relaxation rates. One can also determine the freedom of motion of the probe relative to the rotational diffusion properties of the macromolecule to which it is attached. When held rigidly by the macromolecule, the depolarization of a probe s fluorescence is dominated by the the motion of the macromolecule. 

Another experimental method that has been used to determine orientational correlation functions in macromolecular systems is based on measurements of the time-dependence of the depolarization of fluorescence 26 From these measurements rotational diffusion coefficients and the shape of the rotating macromolecule have been determined.27... 

For chromophores that are part of small molecules, or that are located flexibly on large molecules, the depolarization is complete—i.e., P = 0. A protein of Mr = 25 kDa, however, has a rotational diffusion coefficient such that only limited rotation occurs before emission of fluorescence and only partial depolarization occurs, measured as 1 > P > 0. The depolarization can therefore provide access to the rotational diffusion coefficient and hence the asymmetry and/or degree of expansion of the protein molecule, its state of association, and its major conformational changes. This holds provided that the chromo-phore is firmly bound within the protein and not able to rotate independently. Chromophores can be either intrinsic—e.g., tryptophan—or extrinsic covalently bound fluorophores—e.g., the dansyl (5-dimethylamino-1-naphthalenesulfonyl) group. More detailed information can be obtained from time-resolved measurements of depolarization, in which the kinetics of rotation, rather than the average degree of rotation, are measured. For further details, see Lakowicz (1983) and Campbell and Dwek (1984). 

Rotational diffusion is characterized by the mean square angular deviation during the time interval At (0 ) = GDrAi. Highly anisotropic motion, which is typical for lipid molecules in the membrane, is usually described by two rotational diffusion coefficients Dr and Dri, which correspond to diffusion about the long diffusion axis and perpendicular to it, respectively. The diffusion coefficients are related to corresponding rotational correlation times measured by nuclear magnetic resonance (NMR), electron spin resonance (ESR), fluorescent depolarization, and so on, as ... 

The theory of rotation effects on prolate luminescent molecules in solution and its experimental verification have been developed and compared. Generalized diffusion equations for the rotational motion of an asymmetric rigid motor have been used to given an expression for steady-state fluorescence depolarization. " The radiationless transition from the first excited singlet state of Eosin has been measured by optoacoustic relaxation, and the absolute fluorescence quantum yields of organic dyes in poly(vinyl alcohol) have also been measured by the photoacoustic method. The accuracy of the method has been discussed in the latter paper. Actinometry in flash photolysis experiments has been assisted by new measurements on the extinction coefficient of triplet benzophenone. Matrix-isolation fluorescence spectrometry has been used to detect polycyclic aromatic hydrocarbons from gas chromatography. ... 

A comparison of DODCI fluorescence depolarization in glycerol and ethylene glycol shows the effect of a orientational correlation on excitation transfer effect of rotational diffusion on... 

Fluorescence depolarization time courses contain information about rotational diffusion (26). The main difficulty with streak camera measurements of time-resolved fluorescence depolarization is limited signal-to-noise ratio of single shot records. The fluorescence anisotropy parameter is defined as s... 

The rapid decay of absorption of excited states on the ps time scale has been measured for pyrazolotriazole azomethine dyes. The molecular orientation dynamics of polar dye probes in t-butanol-water mixtures have been determined by ps fluorescence depolarization spectroscopy. Dyes studied in this investigation were the monocations nile blue and thionine, resorufin a monoanion, and nile red a polar but neutral molecule. A very detailed ps study of rotational diffusion of excited states of merocyanine-540 in polar solvents, has also been reported . 

The fluorescence polarization has been observed for the FM and FE bands of pyrene adsorbed on silica gel (9). The excimer fluorescence of pyrene is completely depolarized even in viscous solvents. Rotational diffusion is involved in the process of excimer formation in solutions. The degree of polarization P for the FE fluorescence of adsorbed pyrene has non-zero positive... 

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... 

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. 

Dale and Eisinger have analyzed the effect of rotational mobility Stryer presents an analysis of the errors introduced by assuming = 2/3 and van der Meer et al. present a treatment on the effects of restricted rotational and translational diffusion. Experimentally, one determines the rotational mobility of the dyes by a steady-state or time-resolved fluorescence depolarization experiment. ... 

Rotational diffusion of fluorophores is a dominant cause of fluorescence depolarization. This mode of depolarization is described in the simplest case for spherical rotors... 

Perrin, F., Brownian movement of an ellipsoid. 11. Free rotation and depolarization of fluorescence. Translation and diffusion of ellipsoidal molecules, /. Phys. Radium, 7,1,1936. 

Several phenomena can render the measured anisotropy to values lower than the maximum achievable theoretical values. The most common cause is diffusion of a macromolecule to which the fluorophore is attached. Such rotational diffusion occurs during the lifetime of the excited state and displaces the emission dipole of the fluorophore. Measurement of this parameter provides information regarding the relative angular displacement of the fluorophore between the times of absorption and emission. In fluid solution, most fluorophores rotate extensively in 50-100ps. Hence, the molecules can rotate many times during the typical 1-10 ns excited-state fluorescence lifetime, and the orientation of the polarized emission easily becomes randomized or depolarized. For... 

---