Amino acids, aromatic, phosphorescence

The aromatic amino acids each have two major absorption bands in the wavelength region between 200 and 300 nm (see reviews by Beaven and Holiday(13) and Wetlaufer(14). The lower energy band occurs near 280 nm for tryptophan, 277 nm for tyrosine, and 258 nm for phenylalanine, and the extinction coefficients at these wavelengths are in the ratio 27 7 l.(14) As a result of the spectral distributions and relative extinction coefficients of the aromatic amino acids, tryptophan generally dominates the absorption, fluorescence, and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids. 

The most direct demonstration of triplet-triplet energy transfer between the aromatic amino acids is the ODMR study by Rousslang and Kwiram on the tryptophanyl-tyrosinate dipeptide.(57) Since the first excited singlet state of tyrosinate is at lower energy than that of tryptophan, it is possible to excite tyrosinate preferentially. The phosphorescence of this dipeptide, however, is characteristic of tryptophan, which is consistent with the observation that the triplet state of tyrosinate is at higher energy than that of tryptophan, making tryptophan the expected triplet acceptor. 

Long-lived luminescence from protein-containing materials was reported many years ago. Debye and Edwards reported that a bluish light was emitted from proteins at cryogenic temperatures after illumination/11 Work in the 1950s established the relationship between fluorescence and the long-lived phosphorescence for the aromatic amino acids in proteins/2-41 Konev in his classic work Fluorescence and Phosphorescence of Proteins and Nucleic Acids summarized this early history.1(5)... 

Phosphorescence and photochemistry of aromatic amino acids have been reported.481-483 Triplet states of nucleic acids have also been detected. For example, the phosphorescence of DNA equals the sum of the slow emissions from deoxyadenosine and deoxyguanosine monophosphates present, indicating that only the purine bases phosphoresce.484... 

Luminescence decay curves are also often used to verify that samples do not contain impurities. The absence of impurities can be established if the luminescence decay curve is exponential and if the spectrum does not change with time after pulsed excitation. However, in some cases, the luminescence decay curve can be nonexponential even if all of the luminescing solutes are chemically identical. This occurs for molecules with luminescence lifetimes that depend upon the local environment. In an amorphous matrix, there is a variation in solute luminescence lifetimes. Therefore, the luminescence decay curve can be used as a measure of the interaction of the solute with the solvent and as a probe of the micro-environment. Nag-Chaudhuri and Augenstein (10) used this technique in their studies of the phosphorescence of amino acids and proteins, and we have used it to study the effects of polymer matrices on the phosphorescence of aromatic hydrocarbons (ll). 

The role of the triplet state in biological and biochemical systems continues to receive wide attention. Photoexcited triplet states of prophins and derivatives (389), prophyrins (390), aromatic amino acids (390), aromatic amines (391), and monoanionic thymine (392) have been observed at 77°K. Shiga and Piette (393) confirmed the interpretation of ESR data on triplet-state excitation in proteins by a simultaneous phosphorescence study. 

The environmental sensitivity of the fluorescence and phosphorescence of phenylalanine, tryptophan and tyrosine, and their side chains, is often examined when considering the macromolecular luminescence of natural peptides and proteins. Therefore, lower-lying singlet and triplet states of toluene, aniline and phenol have been extensively studied as the simplest models of the proteins mentioned above, respectively. Knowledge of the various aspects of electronic spectra of the corresponding aromatic amino acids is often exploited to probe those of the proteins137. In other words, accurate information on both... 

Debye and Edwards noted two components in the decay of low temperature protein phosphorescence. The greater part of the decay was exponential in character having lifetimes on the order of several seconds. A weak, and much longer-lived component, however, was reported to have the same emission spectrum but a non-exponential decay. Debye and Edwards claimed that this emission was a result of radical recombination following photoejection of an aromatic amino acid electron into... 

It should be pointed out before concluding this section, that although observed phosphorescence emission from native proteins is almost always characteristic of emission from the aromatic amino acids, a number of proteins have other unsaturated molecules incorporated into their native structure. Molecules such as heme, flavins and carotenoids when complexed with protein contribute to the near UV and visible absorption of the complex and may be involved in energy transfer with aromatic amino acids. Further, triplet probes can often be bound to specific sites in a protein [e.g. inhibitor molecules bound to the catalytic site of an enz5mie) to study such interesting problems as interactions of the probe with metal ions 18) (which may be present in the catalytic site) or with the aromatic amino acids i .20) Since such probes are not native to the protein they are not considered further in this report. 

Several authors have presented data on the quantum yields of fluorescence and phosphorescence of the aromatic amino acids and related compounds as functions of pH 26,3i,42-44) xhe quantum yields are found, in general, to be constant over the pH range of 3—8 but to vary quite dramatically at the extremes of pH. The relative phosphorescence intensities of tyrosine and tryptophan at 77 °K as functions of pH are reproduced in Fig. 5. The pH values where the phosphorescence intensities begin to vary are dose to the pK s for ionization of the amino acids (Table 2). However, in interpreting these results it should be... 

The emission spectra from powders and crystal suspensions of the aromatic amino acids, however, are quite different from those shown in Figs. 3 and 4. The fluorescence and phosphorescence maxima of the powders are red shifted in each case by up to 50 nm and the phosphorescence lifetimes at 77 K are found to be 1.5 sec for tryptophan, 0.4 sec for t5Tosine and 0.5 sec for phenylalanine The phosphorescence lifetimes decrease markedly as the sample temperature increases In the powders the phosphorescence to fluorescence ratios are considerably smaller than in frozen aqueous solutions. The crystal suspensions at room temperature show even greater red shifts in the phosphorescence maxima, with no fine structure observed and with lifetimes on the order of 0.2 sec for each aromatic amino acid >. Although these results along with the phosphorescence excitation spectra bear little resemblence to those expected for the aromatic amino acids, Bogach et al. point out the similarities to phosphorescence and excitation spectra of photoproducts formed in solutions of the aromatic amino acids at low temperatures... 

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. 

Among the first molecules for which triplet state EPR signals were detected in randomly oriented frozen solutions were indole 3) and tryptophan The aromatic amino acids are well suited to triplet EPR studies by virtue of their long-lived phosphorescent states which allow large steady-state triplet populations to be achieved with continuous optical pumping. The triplet states of the aromatic amino acids and... 

In considering the ODMR results of such long-lived phosphorescent species as the aromatic amino acids it is likely that spin-lattice relaxation is not negligible even at the lowest temperatures achieved in any ODMR experiments to date (1.1—1.2 "K). The fast-passage ODMR method and alternative techniques mentioned in the preceding section all have assumed that spin-lattice relaxation between the triplet sublevels could be neglected. Very recently, however, Zuclich et ail. have presented a method for the determination of spin-lattice relaxation rates and for calculating their effects on ODMR responses. 

The total emission and phosphorescence spectra of homopolymers of the three aromatic amino acids are reproduced in Fig. 11. The spectra are recorded at 77 K using diglyme as a solvent tis), x e luminescence properties are summarized in Table 5. Comparisons can be made with the data for the free aromatic amino acids in Table 1 in analyzing the effects of incorporating the amino acids into the homopolymers which have well established regular three dimensional conformations H7,ii8) ... 

Fig. 11. Total emission and phosphorescence spectra of aromatic polyamino acids in diglyme solvent at 77 °K. Amino acid concentration is 5 x 10 M. Total emission spectra (A) poly-L-phenylalanine (10 mv) (B) poly-L-tyrosine (100 mv) (C) poly-L-tryptophan(200mv).Phosphorescence spectra (D) poly-t-phenylalanine (1 mv) (E) poly-L-tr3 tophan (1 mv) (F) poIy-L-tyrosine (10 mv). The amplifier gain setting is given in parentheses. (From Longworth n )).

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