Phosphorescence anisotropy

Dependencies of luminescence bands (both fluorescence and phosphorescence), anisotropy of emission, and its lifetime on a frequency of excitation, when fluorescence is excited at the red edge of absorption spectrum. Panel a of Fig. 5 shows the fluorescence spectra at different excitations for the solutes with the 0-0 transitions close to vI vn, and vra frequencies. Spectral location of all shown fluorescence bands is different and stable in time of experiment and during lifetime of fluorescence (panel b)... 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

Phosphorescence Anisotropy and Rotational Motion 3.4.1. Phosphorescence Anisotropy... 

Experiments involving anisotropy of phosphorescence or of the absorption of the triplet state rely upon the same principles as the measurement of fluorescence anisotropy. All are based upon the photoselection of molecules by polarized light and the randomization of polarization due to Brownian motion occurring on the time scale of the excited state. Anisotropy is defined as... 

Anisotropy of phosphorescence then becomes a powerful tool to study the overall rotation of large biological macromolecules and to study segmental motions which occur in these structures. 

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

Berger and Vanderkooi(88) studied the depolarization of tryptophan from tobacco mosaic virus. The major subunit of the coat protein contains three tryptophans. The phosphorescence decay is non-single-exponential. At 22°C the lifetime of the long component decays with a time constant of 22 ms, and at 3°C the lifetime is 61 ms. The anisotropy decay is clearly not singleexponential and was consistent with the known geometry of the virus. 

The long lifetime of phosphorescence allows it to be used for processes which are slow—on the millisecond to microsecond time scale. Among these processes are the turnover time of enzymes and diffusion of large aggregates or smaller proteins in a restricted environment, such as, for example, proteins in membranes. Phosphorescence anisotropy is one method to study these processes, giving information on rotational diffusion. Quenching by external molecules is another potentially powerful method in this case it can lead to information on tryptophan location and the structural dynamics of the protein. 

G. B. Strambini and W. C. Galley, Detection of slow rotational motions of proteins by steady-state phosphorescence anisotropy, Nature 260, 554-555 (1976). 

H. Kirn and W. C. Galley, Rotational mobility associated with the protein moiety of human serum lipoproteins from tryptophan phosphorescence anisotropy measurements, Can. J. Biochem. Cell Biol. 61, 46-53 (1983). 

G. B. Strambini and E. Gabellieri, Phosphorescence anisotropy of liver alcohol... 

J. W. Berger and J. M. Vanderkooi, Intrinsic phosphorescence anisotropy measurements of the tobacco mosaic virus, work in progress. 

Reticulum ATPase [105,106], Owing to the long-lived nature of the triplet state, Eosin derivatives are suitable to study protein dynamics in the microsecond-millisecond range. Rotational correlation times are obtained by monitoring the time-dependent anisotropy of the probe s phosphorescence [107-112] and/or the recovery of the ground state absorption [113— 118] or fluorescence [119-122], The decay of the anisotropy allows determination of the mobility of the protein chain that cover the binding site and the rotational diffusion of the protein, the latter being a function of the size and shape of the protein, the viscosity of the medium, and the temperature. 

Brown, L.J., Klonis, N., Sawyer, W. H., Fajer. P.G. and Hambly, B.D (2001) Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy, Biochemistry 40, 8283-8291. 

For most proteins, intrinsic triplet lifetimes of aromatics are quite short at room temperatures and thus triplet dye labels must be used. Our own experience has been with the use of triplet probes and triplet anisotropy decay. Since this is somewhat of a new, and we believe under-utilized, field, we will stress it here. Because the triplet yield is usually quite small and either low sensitivity absorption techniques or very low quantum yield phosphorescence measurements must be made, a reasonably high-powered laser is necessary for this kind of experiment. 

Spectroscopies such as UV-visible absorption and phosphorescence and fluorescence detection are routinely used to probe electronic transitions in bulk materials, but they are seldom used to look at the properties of surfaces [72]. As with other optical techniques, one of the main problems here is the lack of surface discrimination, a problem that has sometime been b q)assed by either using thin films of the materials of interest [73, 74], or by using a reflection detection scheme. Modulation of a parameter, such as electric or magnetic fields, stress, or temperature, which affects the optical properties of the sample and detection of the AC component of the signal induced by such periodic changes, can also be used to achieve good surface sensitivity [75]. This latter approach is the basis for techniques such as surface reflectance spectroscopy, reflectance difference spectroscopy/reflectance anisotropy spectroscopy, surface photoadsorption... 

---