Biology Reference
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machinery, where the genetic code is extended to an unnatural fluorescent
amino acid. 25,26 We will briefly present the most common classes of single-
band solvatochromic dyes and examples of their applications.
2.1. Naphthalene sulfonic acid derivatives
One of the first solvatochromic dyes, which still remains an essential tool for
protein and membrane studies, is 1-Anilinonaphthalene-8-Sulfonic Acid
(1,8-ANS). 13,27 In addition to the strong solvent-dependent shift in its
emission spectrum, it shows a dramatic increase in fluorescence intensity
on binding to biomolecules. In bulk water, the fluorescence of this dye is
strongly quenched, while, on being bound to proteins or lipid membranes,
it is efficiently screened from water, resulting in a strong increase in
fluorescence. The other naphthalene sulfonic acid analogues that have
found even more applications in biology are Dansyl derivatives ( Fig. 2.3 ).
The reactive derivative Dansyl chloride was commonly used to label amino
groups in proteins and lipids. Similar to 1,8-ANS, the emission color and
emission intensity of the Dansyl moiety are highly sensitive to solvent
polarity. 28,29 This fluorophore was one of the first used for protein
labeling, but currently it is used only rarely because of its UV absorption
and rather weak solvatochromism.
2.2. Prodan
Prodan is one of the best classical examples of solvatochromic dyes, 17 and has
found numerous biological applications because of its remarkable sensitivity
to solvent polarity together with its relatively small size. Reactive derivatives
of Prodan such as Acrylodan (6-acryloyl-2-dimethylamino-naphthalen) 22,30
or Badan (6-bromoacetyl-2-dimethylaminonaphthalene) 31 have been attached
covalently to proteins via reaction with thiol groups. For instance, Acrylodan
attached to the N-terminus of peptide ligands was used to monitor their
interactions with the holecystokinin receptor. 30 In another representative
study, six cysteine-substituted sites of mouse acetylcholinesterase were labeled
individually with Acrylodan and the kinetics of substrate hydrolysis and
inhibitor binding were examined. While some sites of labeling located far
from the active center did not show any spectral changes of Acrylodan on
inhibitor binding, the sites located near the perimeter of the gorge showed
blue shifts, reflecting the exclusion of solvent and creation of a hydrophobic
environment by the associated ligand. 32 This study is a nice demonstration
how a conformation change upon enzyme-inhibitor interaction can be
specifically “mapped” over all protein. To localize better the fluorophore on
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