You are asked to design an experiment to measure fluorescence from a phenylalani

Question

You are asked to design an experiment to measure fluorescence from a phenylalanine
(Phe) amino acid in a protein that contains one each of Phe, Tyr, and Trp. Use Figure
9.13 and select the best excitation and emission (observation) wavelengths to use.
For your chosen wavelengths, discuss the expected ratio of Phe:Tyr and Phe:Trp fluorescence intensity. Also, discuss the best bandwidths (±??) to use for each wavelength.

8000 6000 0.75 Tyr Trp 4000 0.50 Trp Tyr 0.25 . Phe Phe 220 240 260 280 300 320 250 350 400 Wavelength (nm) Wavelength (nm) FIGURE 9.13 (a) Absorption and (b) fluorescence emission spectra of isolated amino acids, Trp (tryptophan), Tyr (tyrosine), and Phe (phenylalanine). Absorption is plotted as wavelength-dependent absorption coefficient. Fluorescence intensity is in arbitrary units. In proteins fluorescence emission from Tyr is usually less than from Trp. (After Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 65, Springer, New York, NY, 2006.) 9

Explanation / Answer

Tryptophan, tyrosine and phenylalanine are responsible for the inherent fluorescence of proteins, but phenyl alanine is weakly fluorescent and this can be observed in the absence of tryptophan and tyrosine. Excitation wavelengths for Phe (257nm), Tryptophan (280nm) and Tyrosine (274nm) and the emission wavelengths for Phe (282nm), Tryptophan (348nm) and Tyrosine (303nm). The wavelength for best excitation comes around 255nm and emission around 280nm. The bandwidths for phe : Tyr will be 250 to 300 and for emission spectra the band with for Phe :Trp will be 280 to 380. The band widths of 50nm at 250 nm will be the best bandwidths for the wavelength given.

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