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2 Photoconversion of A. victoria GFP
2.1 Discovery and UV/VIS Spectroscopic Investigations
of Photoconversion of GFP
Photoconversion of GFPwas discovered shortly after the cloning of the gfp gene [ 1 , 7 ].
Photoconversion is found to occur in a range of different optical regimes, using UV
and visible light illumination, and with femtosecond and nanosecond pulses as well as
continuous illumination. The report by Chalfie et al. [ 1 ] on recombinant expression of
the gfp gene included a note: “Indeed, the fluorescence produced by 450- to 490-nm
light appeared to be more intense after brief photobleaching by 340- to 390-nm light”
[ 1 ]. A first spectroscopic characterisation of this photochromic reaction appeared in
1995 from the laboratory of Roger Tsien [ 7 ](Fig. 5 ) This paper showed the fluores-
cence changes with UV illumination at 280 nm. Cubitt et al. [ 7 ] proposed photo-
isomerisation as a mechanism and found the spectroscopic changes to be irreversible.
The fluorescence intensity changes in Fig. 5 indicate the occurrence of both photo-
bleaching and photoconversion under the conditions used, considering that at pH 8.0
4 q +5
0 min
3 q +5
13 min
2 q +5
23 min
40 min
0 q +0
Excitation wavelength (nm)
Fig. 5 First spectroscopic demonstration of photoconversion of GFP. Reproduced with permis-
sion from Cubitt et al. [ 7 ]. Selected figure caption: “Behavior of wild-type green fluorescent
protein (GFP) upon progressive irradiation. GFP samples were illuminated at 280 nm from a xenon
lamp and monochromator. Wild-type GFP suffered photoisomerization, decreasing the excitation
amplitude at 395 nm while increasing the amplitude at 475 nm. This effect was not reversible upon
placing the GFP in the dark” [ 7 ]
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