Phosphorescence of Proteins

For phosphorescence measurements, the samples are icalty exdted using a pulsed xenon flash lamp. A commerdally available instrument (the Perkin-Elmer LS-50B luminescence spectrophotometer) is suitable for phosphorescence 

A check on the thoroughness of deoxygenation is provided by the dependence of the phosphorescence lifetime on the amount of oxygen present in the system. Complete removal of O2 fr om a solution of alkaline phosphata.se (10 (jim) 

1 Prepare stock solutions of glucose oxidase (20 mg/ml) and catalase (2 mg/ml) in protein buffer.  

2 To the desired amount of lyophilized protein, add 3 mg of glucose for every ml of final sample volume. Add the desired amount of protein buffer and place the solution in a cuvette. 

3 For eveiy ml of protein/glucose solution, add 1 g. each of the glucose oxidase and catalase stock solutions, stopper the cuvette and mix. 

Gradually, reports appeared about the observation of phosphorescence from proteins at room temperature. Room-temperature phosphorescence of LADH was reported even in the presence of oxygen. However, it turned out that the intense illumin ation of the protein needed to detect the weak phosphorescence apparently caused depletion of oxygen. In ite of the initial confusion, diese results pointed toward the possibility of room-temp ture phosphorescence from proteins. 

Protein Fluorescence maximum (nm) Htosi rescence lifetime (ms) 

At first glance, it appears that oUr undeislantfing of protein phosphorescence is piogressmg fiisler dian past developments in protein fluorescence. However this is the result of die past efforts which clarified many aspects of protein fluorescence and have provided model proteins with previously characterized tryptophan residues. 

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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)... 

S. V. Konev, Fluorescence and Phosphorescence of Proteins and Nucleic Acids, Plenum Press, New York (1967). 

Elsewhere in this volume Millhauser et al. have discussed the application of nitroxide electron paramagnetic resonance (EPR) spin labels to the study of the structure and dynamics of biopolymers. Another type of EPR spin label that also is useful for investigating biopolymer systems is provided by the photoexcited triplet state of an intrinsic chromophore, because a triplet state carries electronic paramagnetism. A major advantage of the photoexcited triplet state of an intrinsic chromophore over an extrinsic spin label such as a nitroxide adduct is the relatively small structural perturbation caused by the former, which consists only of a localized electronic excitation. Although not as widely exploited as fluorescence, the phosphorescence of proteins, originating from the photoexcited triplet state, has received a great deal of attention. EPR afficionados have a natural attraction to photoexcited triplet states that dates back to the... 

Konev, S. V. Fluorescence and phosphorescence of proteins and nucleic acids. 

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

The first example of protein luminescence was made by Beccari in 1746, who detected a visible, blue phosphorescence proceeding from frozen hands when entering a dark room after exposure to sunlight [10]. 

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 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)... 

Essentially nothing is known about tyrosine phosphorescence at ambient temperatures. In frozen solution, tyrosine residues have a phosphorescence decay of seconds. We would expect, however, a decay of milliseconds or shorter at ambient temperature. Observation of tyrosine phosphorescence from proteins in liquid solution will undoubtedly require efficient removal of oxygen. Nevertheless, it could be fruitful to explore ambient temperature measurements, since the phosphorescence decay could extend the range of observation of excited-state dynamics into the microsecond, or even millisecond, time range. 

Although protein phosphorescence was in fact observed earlier than fluorescence, fluorescence of proteins is now widely used, whereas phosphorescence receives much less attention. The reason for this is that until recently it was thought that protein phosphorescence could only be observed in frozen samples, thereby limiting its use. The early literature provides clues that this need not be the case. Beccari reported in 1746 that phosphorescence was observed from a cold hand after it had been exposed to the sunlight/61 A comprehensive coverage of the early sightings of phosphorescence is found in the book by Harvey. 

It is now clear that in the absence of molecular oxygen most proteins phosphoresce in aqueous solutions at ambient temperature.(10) In this chapter we discuss the use of phosphorescence of tryptophan to study proteins, with emphasis on measurements at room temperature. Comparisons between phosphorescence and the more commonly used fluorescence spectroscopy are made. Comprehensive reviews of protein luminescence have been written by Longworth.(n 12 1 A discussion on the use of phosphorescence at room temperature for the study of biological materials was given by Horie and Vanderkooi.(13)... 

A large number of proteins have now been reported to phosphoresce at room temperature. A recent survey of 40 proteins revealed that about 75 % of them showed lifetimes longer than 1 ms.(ro)... 

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. 

From an experimental viewpoint, the wide range of phosphorescence lifetimes is advantageous for the study of proteins. It means that it should be... 

Other groups may cause shortening of the lifetime. The phosphorescence of parvalbumin is quenched by free tryptophan with a quenching rate constant of about 10s M i s l (D. Calhoun, unpublished results). A more extensive survey of proteins or model compounds with known distances between tryptophans is needed to study how adjacent tryptophans affect the lifetime. It should be noted that at low temperature the phosphorescence lifetime of poly-L-tryptophan is about the same as that of die monomer.(12) This does not necessarily mean that in a fluid solution tryptophan-tryptophan interaction could not take place. Thermal fluctuations in the polypeptide chain may transiently produce overlap in the n orbitals between neighboring tryptophans, thus resulting in quenching. 

A survey of the quenching constants for a series of proteins was made using one quencher, nitrite (Table 3.4).(58) It is noted that the phosphorescence lifetime t0 is not correlated with the quenching constant. For example, the... 

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. 

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. 

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. 

M. L. Saviotti and W. C. Galley, Room temperature phosphorescence and the dynamic aspects of protein structure, Proc. Natl. Acad. Sci. U.S.A. 71, 4154-4158 (1974). 

J. Domanus, G. B. Strambini, and W. Galley, Heterogeneity in the thermally-induced quenching of the phosphorescence of multi-tryptophan proteins, Photochem. Photobiol. 34, 15-21 (1980). 

B. Somogyi, J. A. Norman, and A. Rosenberg, Gated quenching of intrinsic fluorescence and phosphorescence of globular proteins, Biophys. J. 50, 55-61 (1986). 

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