Fluorescent Environment-Sensitive Dyes as Reporters of Biomolecular Interactions - Progress in Molecular Biology and Translational Science - page 48

relatively low polarity and hydration, such as, for instance, biological mem-
branes (see below).
On the other hand, the 3HC-B dye is much more appropriate for highly
polar media. Owing to its much weaker electron-donor 2-aryl group
(2-furanyl vs. 4-dialkylaminophenyl in 3HC-A), the dipole moment of its
N* state is relatively low. Therefore, this state cannot be stabilized even
in highly polar aprotic solvents so that its emission is almost negligible
( Fig. 2.6 ). Moreover, in contrast to 3HC-A, the ESIPT inhibition by protic
solvents is not complete so that a clear dual emission depending on solvent
polarity is observed. 53 Thus, the 3HC-B dye is suitable for probing polar
protic environments characterized by high hydration, which corresponds
well to peptides and nucleic acids (see below).
We should note the key differences between single-band solvatochromic
fluorescent dyes and 3HC dyes. While the former shift their emission max-
imum in response to solvent polarity, 3HC dyes may change both the
positions of bands as well as their intensity ratio. This ratio is an additional
channel of spectroscopic information, which allows more detailed (multi-
parametric) characterization of the probe environment. 51 Moreover, as a
result of ESIPT, 3HC dyes are focused in a narrower polarity range, where
they can show higher sensitivity to properties of environment compared to
single-band solvatochromic dyes.

4. APPLICATIONS OF TWO-COLOR DYES FOR
MONITORING BIOMOLECULAR INTERACTIONS
4.1. Monitoring conformational changes of proteins
Conformational changes in proteins can result in significant changes in the
site exposure to bulk water. This idea was validated using a 1-antitrypsin
( a 1-AT), which, during the complex multistep inhibition of proteinases,
undergoes dramatic changes in the tertiary structure ( Fig. 2.7 ). As a 1-AT
contains only one cysteine (Cys-232), this residue was specifically labeled
with a thiol-reactive 3HC-B derivative. 55 The intensity ratio T*/N* of
1.18 for the labeled protein was between that observed for water (0.45)
and for ethanol (2.22), suggesting that the label is partially screened from
bulk water by the protein environment. The interaction of the labeled
a 1-AT with pancreatic elastase led to 65% change in the T*/N* intensity

ratio of the two emission bands, suggesting an increased exposure of the
labeled Cys-232 residue to the bulk water on complex formation. Similar
experiments with elastase and a 1-AT conjugated to a NBD derivative