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398 nm
504 nm
509 nm
476 nm
Fig. 3 Room temperature fluorescence properties of wild-type GFP at pH 8.0. The fluorescence
excitation spectrum measured at 511 nm shows maxima at 280, 398 and 476 nm, the latter two
being assigned to GFP A and GFP B species that have a neutral and anionic p -hydroxybenzylidene-
imidazolidinone chromophore, respectively. The fluorescence emission of the GFP A and GFP B
states is maximum at 509 and 504 nm, respectively. The arrows indicate the Stokes shifts for the
GFP A and GFP B states. The spectra are normalised. The ratio of the excitation maxima at 476 and
398 nm with 511 nm detection is less than the ratio of the corresponding absorption maxima,
reflecting primarily the difference of the fluorescent quantum yields of the GFP A and GFP B states,
which are 0.8 and 0.65, respectively [ 3 ]
timescale to a redshifted species emitting in the green at 508 nm [ 16 ]. This ESPT
reaction was shown to be biphasic in H 2 O, and in D 2 O, with both phases displaying
a kinetic isotope effect (KIE) of about 5 [ 16 ]. The large Stokes shift as well as the
KIE showed that GFP acts as a photoacid, similar to many photoacid compounds
normally studied in solution. GFP is unique to show an ESPT reaction in a protein
environment, and provides an ordered and directional environment for the proton
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