What Affects the Phosphorescence Lifetime

Conceptually, we can separate environmental effects (fcnr) and specific quenching mechanisms (k ), where kpq is due to the presence of a quenching moiety within the protein. Specific quenching effects of externally added quenchers are discussed in Section 3.3.5. 

It has been observed that for some proteins the room temperature phosphorescence lifetimes are increased in D20. The phosphorescence lifetime of liver alcohol dehydrogenase is 300 ms in H2Oand 500 ms in D2O. 10) Phosphorescence lifetimes are often dramatically increased by exchanging hydrogen with deuterium. The reason for this is that decay rates are affected by overtones of the C-H or N-H stretch. In the case of tryptophan in 

Room temperature phosphorescence can be observed from dried proteins. Sheep wool keratin(47) has a phosphorescence lifetime of 1.4 s. Six lyophilized proteins were shown to exhibit phosphorescence at room temperature.(48) The spectra were diffuse, and the lifetime was non-single-exponential, which the authors interpreted as due to inhomogeneous distribution of tryptophans. As the protein was hydrated, the phosphorescence lifetime decreased. This decrease occurred over the same range of hydration where the tryptophan fluorescence becomes depolarized. Hence, these results are consistent with the idea that rigidity of the site contributes to the lifetimes. 

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

In summary, it appears that phosphorescence at room temperature is a function of burial or rigidity of the site, but, as for all excited states, the competing nonradiative pathways are influenced by the polarizability, polarity, and mobility of the local environment. 

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