Access the fluorescence excitation and emission spectra for
phenylalanine, tyrosine, and tryptophan
using the Internet. Answer the following four questions.
a. Estimate peak wavelengths for absorption (at λ> 230nm) and
emission (at λ> 250nm) for each amino
acid.
b. How are the peak absorption wavelengths related to the peak
emission wavelengths? For example, is
the emitted light at lower or higher energy than the absorbed
light? Briefly explain.
c. Which amino acid shows the biggest energy change from absorption
to emission? Explain your
reasoning.
d. Rank these three amino acids from most fluorescent to least
fluorescent. Briefly explain your
reasoning.
a) Peak Wavelength for absorption and emission for each amino acids are:
(i) phenylalanine excitation wavelength = 257 nm
phenylalanine emission wavelength = 282 nm
(ii) Tyrosine excitation wavelength = 274 nm
Tyrosine emission wavelength = 303 nm
(iii) Tryptophan excitation wavelength = 280 nm
Tryptophan emission wavelength = 348 nm
b) Emission wavelength is usually higher than absorption wavelength. In other words, Emission energy is usually lower than absorbed energy. This is because of the fact that fluorescent materials usually under go vibrational relaxation from higher vibrational level(S1,S2,S3) of excited state to lower(or ground state S0) of excited state and from there radiative transition occurs and we see fluorescenc. commonly used term for this process is "Stoke Shift".
c) Tryptophan shows highest energy change from absorption to emission. This is because tryptophan undergo conformational transitions, subunit association, denaturation or substrate binding . Hence affecting the emission spectra more for tryptophan as compared to other two.
d) Most Fluorescent amino acid is tryprophan, second is tyrosin and last comes the phenylanaline. Since tryptophan has greater absorptivity (~5600) than tyrosine (~1400) and phenylalanine (~200), higher quantum yield and resonance energy transfer, the spectrum of tryptophan is most fluorescent among these three.
Access the fluorescence excitation and emission spectra for phenylalanine, tyrosine, and tryptophan using the Internet....
Fluorescence emission spectra and absorption (or excitation) are said to be mirror images of each other. Of the two which has the higher energy spectrum? and Why don’t the two spectra overlap completely?