Tryptophan Phosphorescence Emission from Proteins

It is clear that the wide range of protein phosphorescence lifetimes is due to various specific quenching mechanisms kq) or due to flexibility of the tryptophan site, thereby affecting km. It also follows that phosphorescence will be very sensitive to conformational fluctuations since subtle changes in distance or orientation relative to a specific quenching moiety within the protein will affect the lifetimes dramatically. The phosphorescence emission from protein tryptophan remains relatively unexplored in terms of investigation of dynamic structure-function relationships. 

The fluorescence of purified histones has been studied by several different groups, 90 95) with the most detailed studies being on calf thymus histone HI. Histone HI, which binds to the outside of core particles, contains one tyrosine and no tryptophan. This protein exhibits a substantial increase in fluorescence intensity in going from a denatured to a folded state.<90) Collisional quenching studies indicate that the tyrosine of the folded HI is in a buried environ-ment.(91) Libertini and Small(94) have identified three emissions from this residue when in the unfolded state with peaks near 300, 340, and 400 nm. The 340-nm peak was ascribed to tyrosinate (vide infra), and several possibilities were considered for the 400-nm component, including room temperature phosphorescence, emission of a charge transfer complex, or dityrosine. Dityrosine has the appropriate spectral characteristics, but would require... 

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. 

Figure 3.2 shows the fluorescence and phosphorescence emission spectrum from tobacco mosaic virus coat protein. These spectra are fairly typical of the tryptophan emission spectra observed from proteins at room temperature. 

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-... 

Mazhul et alP have reported that long-lived luminescence could be detected in intact human erythrocytes and white blood cells at ambient temperature. They have shown by emission spectra and pH dependency that this emission arises from tryptophan. The emission was not singleexponential, suggesting that more than one population of tryptophan emitted. Identification of the emitting species has not yet been conclusively made, but the white blood cell protein content is about 10% actin, a protein known to phosphorescence.(91)... 

Bismuto et alF compared the phosphorescence from both tuna and sperm whale apomyoglobin. The emission occurs from a tryptophan in the A helix. The temperature dependence of lifetime and the position of the 0-0 vibrational band differ as a function of temperature for the two proteins. The authors interpreted their results to indicate that the microenvironment of the tryptophan in sperm whale apomyoglobin possesses a higher degree of internal flexibility than that in the tuna protein. 

Denaturation of the protein results in blue shifts of the phosphorescence to match the emission of tr5 ptophan in a similar medium. In the instances of narrow bandwiths mentioned above, denaturation b dens the phosphorescence peaks to the more commoidy found 200 cm i. Thus, the variations noted in the phosphorescence spectra of Class B proteins appear to be due to differing amounts of shielding of the emitting chromophores from the solvent medium. The variations are for the most part removed with denaturation when all tryptophan residues are given roughly equal exposures to the solvent. 

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