Phosphorescence Anisotropy Decays

The information avaikdde from an anisotropy decay is limiled to times during which emission occurs. For this 

Another approach to measuring long correlation times is to use luminescent MLCs. These probes, which display lifetimes rang fixim 100 ns to 10 )ts, have only recendy become avaihide (Chapter 20). They are typically 

It is important to notice that the use of a long-lifetime probe allowed measurement of overall rotation of the 

Rgure 11.22. FD anbotrofigr decays frfRn(b 2(dcbpy)-PE2 ia DPPG vendes. Reprinted from ReC 88, with pennissim from Aeedenoic Press, lae. 

The MLCs have several advantages over the phosphorescent probes. In contrast to phosphorescence, the luminescence from MLCs can be measured in the presence of dissolved oxygen. The MLCs are only partially quenched by ambient oxygen, whereas phosphorescence is usually completely quenched. Additionally, there are relatively few phosphorescent probes, but there are numerous MLCs (Chapter 20). 

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Figure 7. Phosphorescence anisotropy decay of the dodecanoyl-amidoeosin fatty acid probe in DMPC vesicles at 30 C (lower curve) and 10 C (upper curve). The solid lines represent the best fit analyses according to Equation...

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)... 

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. 

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. 

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. 

Figure 1. Delayed Luminescence Anisotropy (DIA). A) The sample (lined sphere) is excited with polarized light (vector. The emission is observed at 90" through an analyzer set at an angle corresponding to vector %). B) The ground state G is excited to the singlet state S (square) which decays directly (fluorescence f) or indirectly (phosphorescence p or delayed fluorescence by thermal reactivation to S) through the triplet state T. The circled states are long-lived and the hollow line connecting T and G denotes a slow decay process. Non-radiative modes are omitted (see text).

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