DF445 ¼ F445
280 F445
300 ð1Þ
where F445
280 represents the fluorescence intensity at 445 nm under
280 nm excitation, F445
300 is the fluorescence intensity at 445 nm
under 300 nm excitation. DF445 is contributed mainly by the
intra-molecular energy resonance between OPA and the aromatic
side chain of amino acids. Basically all amino acids except tyrosine
and tryptophan show an emission plateau around 290 nm (Fig. 2c),
so DF445 can be considered as a combined FRET signal from tyrosine
and tryptophan for an amino acid mixture. Although the contribution
of tyrosine on DF445 is ca. 37% of the same concentration of
tryptophan (data from Fig. 2), the natural abundance of tyrosine is
at least 3 times higher than tryptophan, which may cause problems
if tryptophan content is the major concern of our study.
Although somewhat decreased in fluorescence intensity, the
interference from co-existing tyrosine can be further reduced by
red-shifting the emission wavelength from 445 to 475 nm if
necessary