Tryptophan phosphorescence

In the case of carboxypeptidase B, Shaklai et al.(2lT> compared the relative contributions to the protein phosphorescence from tyrosine and tryptophan for the apoenzyme, the zinc-containing metalloenzyme in the absence of substrate, the metalloenzyme in the presence of the substrate iV-acetyl-L-arginine, and the metalloenzyme in the presence of the specific inhibitor L-arginine. The tyrosine tryptophan emission ratio of the metalloenzyme was about a factor of four smaller than that of the apoenzyme. Binding of either the substrate or the inhibitor led to an increase in the emission ratio to a value similar to that of the apoenzyme. The change in the tyrosine tryptophan phosphorescence ratio was attributed to an interaction between a tyrosine and the catalytically essential zinc. The emission ratio was also studied as a function of pH. The titration data are difficult to interpret, however, because a Tris buffer was used and the ionization of Tris is strongly temperature dependent. In general, the use of Tris buffers for phosphorescence studies should be avoided. 

J. Zuclich, D. Schweitzer, and A. H. Maki, Optically detected magnetic resonance of the tryptophan phosphorescent state in proteins, Photochem. Photobiol. 18, 161-168 (1973). 

Tryptophan Phosphorescence from Proteins at Room Temperature... 

The range of six orders of magnitude for lifetimes of tryptophan phosphorescence in proteins at room temperature is larger than for fluorescence. The lower limit for fluorescence lifetime is about 0.5 ns, while the upper limit is 8 ns.(21> Typical values range from 3 to 5 ns. 

Dorn anus el al.(74> proposed that the ratio of phosphorescence intensity to lifetime, P/t), of tryptophan phosphorescence as a function of temperature be used to distinguish heterogeneity in emission from multitryptophan proteins. Since different tryptophans within one protein show different temperature-... 

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. 

The long-lived phosphorescence of the tryptophan in alkaline phosphatase is unusual. Horie and Vanderkooi examined whether its phosphorescence could be detected in E. coli strains which are rich in alkaline phosphatase.(89) They observed phosphorescence at 20°C with a lifetime of 1.3 s, which is comparable to the lifetime of purified alkaline phosphatase (1.4 s). Long-lived luminescence was not observed from strains deficient in alkaline phosphatase. The temperature dependence of tryptophan phosphorescence in the living cells was slightly different from that for the purified enzyme, indicating an environmental effect. 

Phosphorescence is readily detectable from most types of proteins at room temperature. Tryptophan phosphorescence lifetimes and yields are very sensitive to environment, and therefore phosphorescence is sensitive to conformational changes in proteins. Fundamental questions concerning exactly what parameters affect lifetime and spectra of tryptophan in proteins remain still to be answered. 

N. Barboy and J. Feitelson, Quenching of tryptophan phosphorescence in alcohol dehydrogenase from horse liver and its temperature dependence, Photochem. Photobiol. 41, 9-13 (1985). 

J. M. Vanderkooi, S. Papp, T. Samoriski, S. Pikula, and A. Martonosi, Tryptophan phosphorescence of the Ca2 + -ATPase of sarcoplamic reticulum, Biochim. Biophys. Acta 957, 230-236(1988). 

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). 

V. M. Mazhul, Y. S. Ermolaev, and C. V. Konev, Tryptophan phosphorescence at room temperature New method for the study of the structural composition of biological membranes and proteins in cells, Zh. Prikl. Spectrosk. 32, 903-907 (1980). 

Quantum Yield Efficiency of fluorescence percentage of incident energy emitted after absorption. The higher the quantum yield, the greater the intensity of the fluorescence, luminescence, or phosphorescence. See Papp, S. and Vanderkooi, J.M., Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics, Photochem. Photobiol. 49, 775-784, 1989 Plasek, J. and Sigler, K Slow fluorescent indicators of membrane potential a survey of different approaches to probe response analysis, J. Photochem. Photobiol. 33, 101-124, 1996 Vladimirov, Y.A., Free radicals in primary photobiological processes, Membr. Cell Biol. 12, 645-663, 1998 Maeda, M., New label enzymes for bioluminescent enzyme immunoassay, J. Pharm. Biomed. Anal. 30, 1725-1734, 2003 Imahori, H., Porphyrin-fullerene linked systems as artificial photosynthetic mimics, Org. Biomol. Chem. 2, 1425-1433, 2004 Katerinopoulos, H.E., The coumarin moiety as chromophore of fluorescent ion indicators in biological systems, Curr. Pharm. Des. 10, 3835-3852, 2004. 

Strambini and Gabellieri (1984) found the tryptophan phosphorescence of lysozyme and several other proteins to have similar long lifetimes (about 1 sec) in the dry state. In solution protein phosphorescence lifetimes are generally widely different and short. The long dry-state... 

As we have mentioned previously, the phosphorescence of aromatic amino acids in solution is completely quenched at room temperature by rapid non-radiative processes. Non-radiative processes appear to be practically dormant at 77 °K since the sum of the fluorescence and phosphorescence quantum yields is close to unity for each aromatic amino acid (Table 1). As the temperature is raised the phosphorescence quantum yield begins to decrease drastically as solvent reorientation sets in. This occurs between 170° and 200 °K for frozen 0.5% glucose solutions A plot of the variation of the tryptophan phosphorescence quantum yield with temperature is shown in Fig. 7. 

At alkaline pH the fluorescence of Class B proteins is found to be that of tyrosinate isi-i ). A report of Vladimirov and Zimina )that the fluorescence of serum and egg albumins at pH 13 is entirely due to tryptophan is probably in error the observed luminescence is most likely that of tyrosinate. Tryptophan and t3n-osinate fluorescence spectra are quite similar and hfetime measurements are sometimes necessary for definite identification. The phosphorescence of Class B proteins at alkaline pH generally has a considerable tr5 tophan component along with a dominant tyrosinate contribution 26,164). Thus, trp- -t T < > transfer appears to be very efficient at the singlet level and enhanced intersystem crossing to the tryptophan triplet at high pH 26) also contributes to the tryptophan fluorescence quenching and to the production of tryptophan phosphorescence of Class B proteins at high pH. It is possible that tyr( ) - -trp triplet transfer also occurs to an extent in some proteins. 

Sdwnerte, I. A.. Sled. D. G., and Gafhi, A., 1997, Time resolved room tenycaatuie tryptophan phosphorescence in proteins. Medh oifrfiiOmoL27t 49-71. 

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