Proteins contain three aromatic amino acid residues (tryptophan, tyrosine, phenylalanine)

which may contribute to their intrinsic fluorescence. Cofactors such as FMN, FAD, NAD and
porphyrins also exhibit fluorescence.

The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic
residues. Protein fluorescence is generally excited at 280 nm or at longer wavelengths, usually
at 295 nm. Most of the emissions are due to excitation of tryptophan residues, with a few
emissions due to tyrosine and phenylalanine.

The three residues have distinct absorption and emission wavelengths. They differ greatly in
their quantum yields and lifetimes. Due to these differences and to resonance energy transfer
from proximal phenylalanine to tyrosine and from tyrosine to tryptophan, the fluorescence
spectrum of a protein containing the three residues usually resembles that of tryptophan.

Tryptophan has much stronger fluorescence and higher quantum yield than the other two
aromatic amino acids. The intensity, quantum yield, and wavelength of maximum fluorescence
emission of tryptophane is very solvent dependent. The fluorescence spectrum shifts to
shorter wavelength and the intensity of the fluorescence increases as the polarity of the
solvent surrounding the tryptophane residue decreases. Tryptophan residues which are buried
in the hydrophobic core of proteins can have spectra which are shifted by 10 to 20 nm
compared to tryptophans on the surface of the protein. Tryptophan fluorescence can be
quenched by neighbouring protonated acidic groups such as Asp or Glu.

Tyrosine, like tryptophan, has strong absorption bands at 280 nm, and when excited by light
at this wavelength, has characteristic emission profile. Tyrosine is a weaker emitter than
tryptophan, but it may still contribute significantly to protein fluorescence because it usually
present in larger numbers. The fluorescence from tyrosine can be easily quenched by nearby
tryptophan residues because of energy transfer effects. Also, tyrosine can undergo an excited
state ionization which may result in the loss of the proton on the aromatic hydroxyl group that
leads to quenching of tyrosine fluorescence.

Phenylalanine with only a benzene ring and a methylene group is weakly fluorescent. The
experimental sensitivity (the product of quantum yield and molar absorbtivity maximum) is
especially low for this residue. Phenylalanine fluorescence is observed only in the absence of
both tyrosine and tryptophane. The simple structure of phenylalanine may preeminently
demonstrate the effect of structure on fluorescence. Adding a hydroxyl group, as in tyrosine,
causes a 20 fold increase in fluorescence. If an indole ring is added as in tryptophan, the
relative fluorescence increases to 200 times that of phenylalanine.

and cys cannot produce such type of fluorescence