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measures of mRNA and proteomic screens give some information about
specific enzyme levels, many enzymes are activated during posttranslational
processing. Thus, a more direct measure is needed to discern actual enzyme
activity. Fluorescent indicators are uniquely suitable for this task because
of their ability to undergo rapid (milliseconds to seconds) transition from
a “silent” nonfluorescing to a fluorescing state, or from a shorter to a longer
emission wavelength. The goal of designing an enzyme-sensitive probe is to
link the change in the fluorescence state to the activity of the enzyme, and
this has been accomplished over the years using a number of different mac-
romolecular chemistries and configurations. In some configurations, a single
enzyme molecule can activate multiple fluorophore-linked substrates within
the same microenvironment and thereby produce an amplification of the
fluorescent signal in the diseased tissue (the basis for use of enzymes such
as
-lactamase, etc., as reporter genes). As we discuss below,
a number of different chemistries have been used in the development of
fluorescent probes targeting different diseases/enzyme systems. The overall
difference is in the way the fluorescence-generating substrate is targeted to
the enzyme (e.g., covalently vs. noncovalently) and the way in which the
precursor (substrate) molecule is made silent (quenched) in the nonactivated
state. For example, fluorophores may be quenched by other molecules as a
result of collisional interaction (dynamic quenching), by interaction with
surface plasmons (such as a gold nanoparticle surface), or by closely posi-
tioned secondary fluorophores that quench emission because of formation
of noncovalent complexes (close quenching). The efficacy of FRET is
determined by the energy transfer rate, which is a function of the overlap
of the donor emission and acceptor excitation spectra, the relative orienta-
tion of the transition dipoles, and the distance between the donor and the
acceptor.
60
The latter is usually measured in Forster's distance which is usu-
ally limited to 10 nm.
61
Fluorophores can also be switched on and off by
modulation of their electronic properties through oxidation-reduction
and other chemical reactions.
62
b
-galactosidase,
b
5. MACROMOLECULAR FLUORESCENT PROBES
The most frequently used amino-reactive fluorescent dye FITC
(fluorescein isothiocyanate) reacts both with
a
-N-terminal amino groups
and N-
-amino groups of lysine at high pH values with the formation
of the corresponding thiocarbamylates.
63
This fluorochrome-activated ana-
log is frequently used for tagging proteins with readily detectable green
e
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