TYROSINE FLUORESCENCE IN PROTEINS
Authors: Edelhoch Harold , Perlman Robert L. , Wilchek Meir 
Published: January 2007

The problem of observing tyrosine emission in proteins containing tryptophan is related to both the absorption and emission properties of these two amino acids. First, the absorption of tryptophan is several times larger than tyrosine at all wavelengths. In the two compounds, acetyl tryptophan‐amide and acetyl tyrosinamide, the ratio of the molar extinction coefficients of the latter to the former is maximal at 274 mμ and 232 mμ (Figure 11). At 270 mμ, the wavelength used for excitation in most experiments, the ratio is 0.235. The 270 mμ wavelength was chosen instead of 274 mμ in order to eliminate more of the scatter peak of the exciting radiation. The second problem is that the quantum yield of tyrosine is smaller than that of tryptophan in many proteins, 20 polypeptides21,22, 28 and small peptides.20 Due to these two factors, a very high fraction of the total emission is due to tryptophan. Of major importance in observing the tyrosine band is the wavelength peak of tryptophan emission. The smaller the latter, the greater the overlap and the less likely that the tyrosine band will be resolved as a shoulder or separate peak. In the absence of its resolution, the contribution of tyrosine to an emission band may be shown by exciting at two wavelength, where the ratio of absorption of tryptophan to tyrosine is high and low. In this way, the tyrosyl emission in bovine serum albumin34 and ovalbumin35 has been detected. Another method is to conjugate a dye which quenches tryptophan but not tyrosine emission by energy transfer. This result has been accomplished with the dimethylamino naphthalene sulfonyl conjugate of human serum albumin. It has shown that phosphate preferentially quenches tyrosine emission.20,36 Thus, if tyrosyl residues are accessible to phosphate their emission may be verified. It may also be possible to selectively eliminate tryptophan emission by chemical modification, as with N‐bromosuccinimide. In chemical changes the product may, however, quench tyrosyl or tryptophanyl fluorescence by energy transfer or other mechanisms.

There is considerable physical and chemical data on the two macromolecules, i.e., nuclease4 and bovine growth hormone,32,33 which reveal them to be highly organized structures and consequently proteins. In the case of the two hormones, PTH and ACTH analog, very little relevant data exists concerning their structure. It is instructive to compare the fluorescence behavior of the two proteins with that of the two hormones, i.e., PTH and ACTH analog.

Acidification of the nuclease or bovine growth hormone produces major changes in the quantum yields of both chromophores. Very small, inconsequential changes were observed with PTH and ACTH analog. In accord with their behavior in acid, the temperature dependence of the former two proteins was very abnormal, whereas in the latter two hormones it was similar to that of the amino acid models. Moreover, the tryptophanyl emission peaks of PTH and ACTH analog occurred near 350 mμ and, therefore, at longer wavelengths than that of the nuclease and bovine growth hormone. Guanidine or dioxane in the case of the nuclease, or urea with bovine growth hormone, strongly modified their fluorescence spectra. In contrast, guanidine (5M) had very little effect on the fluorescence spectra of PTH or ACTH analog at neutral pH. We have observed that guanidine (5M) reduces the fluorescence of either N‐acetyl tryptophanamide or N‐acetyl tyrosinamide by only a few percent. It is evident that the tyrosyl and tryptophanyl residues in the two proteins are involved in interactions which have profound effects on their emissive properties. In the case of PTH and ACTH analog the two chromophores appear to be free and exposed to the solvent. From their fluorescence behavior under a variety of conditions, it appears likely that these two hormones have very little, if any, tertiary structure. One would expect that tyrosyl and tryptophanyl residues, because of their relatively low polarity, would be interior residues if the molecule had a globular form. It is, of course, dangerous to extrapolate from the properties of only two types of residues to that of the whole molecule. However, in the globular proteins whose structures have been determined by x‐ray analysis, most amino acids have fixed coordinates.37,38 It seems unlikely, therefore, that two bulky residues that can form strong hydrophobic interactions will be free and the remainder fixed.

The only data which might indicate that PTH has structure are the differences in tryptophan quenching in water compared to guanidine due to energy transfer to ionized tyrosine. This result can be explained if PTH is more extended in guanidine than in water. It is known that concentrated guanidine solutions are better solvents than water for proteins.39 Consequently, proteins or polypeptides would be expected to have a greater effective volume or root mean square end‐to‐end distance in guanidine.40

There is chemical data which indicates that the tryptophan and the two methionine groups in PTH are free. The latter are readily oxidized, and tryptophan reacts with both N‐bromosuccinimide and 2‐hydroxy‐5‐nitrobenzyl bromide.3

If either PTH or ACTH analog possesses structure, it would have to be formed from a less structured molecule that existed at the time of extraction in the former or synthesis in the latter. The methods of extraction and purification would disorganize any ordered structure that PTH may have in situ. It is extracted in 90% phenol and then distributed between n‐butanol and 20% acetic acid in counter current distribution.1,2 In aqueous solutions at neutral pH it might, however, recover its structure. Its remarkable stability survives brief periods of boiling in 0.1M HCl or hot 80% acetic acid, treatment with guanidine, urea and trichloracetic acid.