Fluorescence of Tyrosine

Tyrosine fluorescence emission in proteins and polypeptides usually has a maximum between 303 and 305 nm, the same as that for tyrosine in solution. Compared to the Stokes shift for tryptophan fluorescence, that for tyrosine appears to be relatively insensitive to the local environment, although neighboring residues do have a strong effect on the emission intensity. While it is possible for a tyrosine residue in a protein to have a higher quantum yield than that of model compounds in water, for example, if the phenol side chain is shielded from solvent and the local environment contains no proton acceptors, many intra- and intermolecular interactions result in a reduction of the quantum yield. As discussed below, this is evident from metal- and ionbinding data, from pH titration data, and from comparisons of the spectral characteristics of tyrosine in native and denatured proteins. 

Time-dependent fluorescence measurements have been made on tyrosine in calf thymus nucleosome core particles by Ashikawa et al. S7) Based on the salt dependence of the decay data, the tyrosines were divided into two classes. At 20 to 400 mM salt, about half of the tyrosine residues appear to be partially quenched, possibly by resonance energy transfer to DNA bases. The other half are thought to be statically quenched, possibly by hydrogen bonds this quenching is partially eliminated at about 2 M salt. In view of the number of tyrosines per nucleosome core particle (estimated at 30), it is impossible to make a more detailed analysis of the decay data. 

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 

Histone HI from the fruit fly Ceratitis capitata has two tyrosine residues. Jordano et al.( )2 have observed two differences from calf thymus HI (1) the apparent quantum yield does not increase on protein folding and (2) there is a pH- and conformation-dependent shoulder at 340 nm in the emission spectrum. This group has attributed this 340-nm emission to tyrosinate.(97) Their studies demonstrate that the folding of histone HI from C. capitata is pH and ionic strength dependent. The possibility of tyrosinate formation at neutral pH is discussed in greater detail in Section 1.5.2. 

The H2a-H2b histone dimer also has strong salt-dependent conformational properties, with a transition near 0.5 M NaCl.(93) Above 0.5 M NaCl, the tyrosine fluorescence emission becomes less quenchable by Cs+, and the dimer structure becomes more compact. 

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J. L. Comog and W. R. Adams, The fluorescence of tyrosine in alkaline solution, Biochim. 

R7. Fluorescence of tyrosine in protein is generally quenched as a result of the tertiary structure of the protein. A structural modification of the protein would affect the fluorescence properties of the protein, including those of the tyrosine. Thus, in some cases, one can observe an increase in the fluorescence intensity at 303 nm and the quantum yield of the tyrosine and a decrease in its anisotropy. 

Natural fluorescent labeling of proteins is derived from their primary structure, i.e. mainly from the type, number and occurrence of amino acids having fluorescent properties. For native (intrinsic) fluorescence of proteins tryptophan and tyrosine are specially responsible, although some other amino acids (phenylalanine, histidine, arginine) are fluorescent, too. The fluorescence contribution of these other amino acids is, however, extremely The fluorescence of tyrosine is normally... 

Model compound and difference spectral studies have been carried out on tyrosine and related compounds by Wetlaufer (1956), Laskowski (1957), Edelhoch (1958), Chervenka (1959), Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Foss (1961) (see also Section VI,Z)). Studies of the fluorescence of tyrosine—activation spectrum, fluorescence emission spectrum, and quantum yield—have been reported by Teale and Weber (1957). The pH-dependence of the fluorescence of tyrosine and related compounds was studied by White (1959) fluorescence-polarization studies from the same laboratory were reported by Weber (1960a) for simple compounds, and for proteins (Weber, 1960b). Teale (1960) has carried out extensive studies on the fluorescence characteristics of a score of proteins. 

Kierdaszuk, B., Gryc27nski, I , Modrak Wojcik, A., Bzowska, A., Shugar, D , and Lakowicz, J. R. (1995) fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation. Photochemistry and Photobioiogy. 61 319-324. 

Proteins which contain tryptophan fluoresce at about 350 nm when excited at 270-305 nm. The fluorescence of tyrosine at 305 nm is not visible in LDH this is normal for proteins which also contain tryptophan 223). The fluorescence of tryptophan residues in LDH is much higher in the native apoprotein than in the unfolded protein or its alkaline hydrolysate... 

Other observations relevant to possible ligands to the Cu2+ are (a) None of the plastocyanins analyzed contains tryptophan or arginine, (b) The fluorescence of tyrosine in parsley plastocyanin is partially quenched but there is no evidence that t5nosine is bound to Cu2+. (c) Spinach and French bean plastocyanin contain two histidine residues while parsley plastocyanin appears to have only one. 

Important structural transitions occur in extreme pH values, i.e., below pH 4.0 and above pH 10.5, The reversibility of the transition which occurs between pH 3 and 4 was demonstrated by following different parameters (e.g., fluorescence of the unique tryptophan, fluorescence of tyrosine, tyrosine absorption, molar ellipticity, and reduced viscosity). The same transition curve is observed with all parameters, with a midpoint corresponding to pH 3.9 (Fig. 5.5). 

Kojima, S., et al. (1998). Fluorescent properties of model chromophores of tyrosine-66 substitute mutants of Aequorea green fluorescent protein (GFP). Tetrahedron Lett. 39 5239-5242. 

The synthesized CPMV-alkyne 42 was subjected to the CuAAC reaction with 38. Due to the strong fluorescence of the cycloaddition product 43 as low as 0.5 nM, it could be detected without the interference of starting materials. TMV was initially subjected to an electrophilic substitution reaction at the ortho-position of the phenol ring of tyrosine-139 residues with diazonium salts to insert the alkyne functionality, giving derivative 44 [100]. The sequential CuAAC reaction was achieved with greatest efficiency yielding compound 45, and it was found that the TMV remained intact and stable throughout the reaction. 

Sakura, S. and Fujimoto, D. (1984) Absorbtion and fluorescence studies of tyrosine derived crosslinking amino acids from collagen. Photochemistry and Photobiology 40, 731-734. 

The pK of tyrosine explains the absence of measurable excited-state proton transfer in water. The pK is the negative logarithm of the ratio of the deprotonation and the bimolecular reprotonation rates. Since reprotonation is diffusion-controlled, this rate will be the same for tyrosine and 2-naphthol. The difference of nearly two in their respective pK values means that the excited-state deprotonation rate of tyrosine is nearly two orders of magnitude slower than that of 2-naphthol.(26) This means that the rate of excited-state proton transfer by tyrosine to water is on the order of 105s 1. With a fluorescence lifetime near 3 ns for tyrosine, the combined rates for radiative and nonradiative processes approach 109s-1. Thus, the proton transfer reaction is too slow to compete effectively with the other deactivation pathways. 

The fluorescence decay parameters of tyrosine and several tyrosine analogues at neutral pH are listed in Table 1.2. Tyrosine zwitterion and analogues with an ionized a-carboxyl group exhibit monoexponential decay kinetics. Conversion of the a-carboxyl group to the corresponding amide results in a fluorescence intensity decay that requires at least a double exponential to fit the data. While not shown in Table 1.2, protonation of the carboxyl group also results in complex decay kinetics.(38)... 

Cowgill pointed out that there are essentially two distinct quenching processes of tyrosine fluorescence resulting from association with the peptide bond.(3) Tyrosines affected by these mechanisms are classified in Table 1.3 as... 

Eisinger(55) also noted that it is difficult to obtain accurate data with phenylalanine as the donor and either tryptophan or tyrosine as the acceptor. The source of this problem is the weak S, - S0 absorption of phenylalanine compared to that of tyrosine or tryptophan, which leads to considerable experimental uncertainty in measuring the sensitized acceptor emission. This error may account for the finding of Kupryszewska et al.<56> that the sensitization of the acceptor fluorescence was less than the quenching of the donor fluorescence in their study of phenylalanine-to-tyrosine energy transfer... 

By comparing time-resolved and steady-state fluorescence parameters, Ross et alm> have shown that in oxytocin, a lactation and uterine contraction hormone in mammals, the internal disulfide bridge quenches the fluorescence of the single tyrosine by a static mechanism. The quenching complex was attributed to an interaction between one C — tyrosine rotamer and the disulfide bond. Swadesh et al.(()<>> have studied the dithiothreitol quenching of the six tyrosine residues in ribonuclease A. They carefully examined the steady-state criteria that are useful for distinguishing pure static from pure dynamic quenching by consideration of the Smoluchowski equation(70) for the diffusion-controlled bimolecular rate constant k0,... 

In testing the possibility of proton transfer as a quenching mechanism of tyrosine in oligopeptide/polynucleotide complexes, Brun et a/.(102) compared the fluorescence emission spectra of the tyrosine and O-methyltyrosine tripeptides. They noted that, in the complex, the O-methyltyrosine tripeptide had a unique secondary emission near 410 nm. Whether this emission is related to that observed by Libertini and Small(94) is an important question. While one must consider the possibility that two tyrosine side chains could be converted to dityrosine, (96) which has a fluorescence at 400 nm, another intriguing possibility is ambient temperature tyrosine phosphorescence. This could happen if the tyrosine side chain is in a rigid, protective environment, very effectively shielded from collisions with quenchers, particularly oxygen. 

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