Biology Reference
In-Depth Information
diaphorase, inside living cells yields the fluorescent resorufin ( 62 ), making
this molecule a valuable reagent for the determination of cell viability. 111
Resorufin can also be reduced to a nonfluorescent form as in compound
70 . This molecule is oxidized by H 2 O 2 in a horseradish peroxidase-catalyzed
reaction to yield resorufin and is widely used in the enzyme-linked immu-
nosorbent assay (ELISA). 3,100
The amine-containing phenoxazoles are also useful scaffolds for enzyme
assays. Analogous to the rhodamine-based protease substrates, acylation of
Cresyl Violet and its derivatives with amino acids gives valuable fluorogenic
compounds. 27,104,112,113 One example is compound 71 , which releases a far-
red fluorophore (
10 4 M 1 cm 1 , and
l
max /
l
¼
633/675 nm,
e ¼
3.6
em
0.10) upon cleavage of the peptide. 114 In a similar vein, the Cresyl
Violet sulfonamide 72 has been described as a fluorogenic glutathione
transferase substrate. 91
F ¼
8.3. Photoactivatable probes
Photolabile “caged” resorufin was developed by Mitchison for use in im-
aging assays to determine the dynamic behavior of actin filaments. 99
A benefit of this dye is the relatively long wavelengths (
572/
585 nm) when compared to the caged fluoresceins and rhodamines. How-
ever, like the other alkyl resorufins, the quenching of the fluorescence is
incomplete, limiting the utility of this dye in experiments that require
high-contrast dyes.
l
max /
l
¼
em
8.4. Indicators
The long wavelength of resorufin makes it an attractive platform for sensors.
One example is the reduced boronic ester 74 , which reacts spontaneously
with H 2 O 2 to yield the fluorescent resorufin. Other phenolic fluorophores,
such as fluorescein, can be masked in a similar fashion. 115 Resorufin can also
be appended with arsenical substituents to give the resorufin arsenical hair-
pin binder (ReAsH; 75 ). This compound is nonfluorescent in solution but
becomes fluorescent upon binding to an engineered tetracysteine motif on a
protein. 116 Yet another sensor molecule based on the phenoxazines is Nile
Red ( 8 ). As highlighted in Section 3 , this compound becomes fluorescent in
a lipophilic environment. In addition to staining lipid droplets in cells, 20 this
fluorogenic phenomenon has also been used to perform super-resolution
microscopy on cellular membranes. 117
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