A FRET-based approach to ratiometric fluorescence detection of hydrogen peroxide

Article abstract of DOI:10.1021/ja063308k

We report the synthesis, properties, and biological applications of Ratio-Peroxyfluor-1 (RPF1), a new ratiometric fluorescent reporter for hydrogen peroxide. RPF1 is comprised of a two-fluorophore cassette, where the spectral overlap between coumarin dono

Full text of DOI:10.1021/ja063308k

Published on Web 07/04/2006
A FRET-Based Approach to Ratiometric Fluorescence Detection of Hydrogen
Peroxide
†
‡
,†
Aaron E. Albers, Voytek S. Okreglak, and Christopher J. Chang*
Departments of Chemistry and Molecular and Cell Biology, UniVersity of California, Berkeley, California 94720
Received May 11, 2006; E-mail: chrischang@berkeley.edu
Hydrogen peroxide is an essential oxygen metabolite in living
systems, and mounting evidence supports its role as a messenger
in cellular signal transduction.1 This byproduct of aerobic respira-
Scheme 1. Synthesis and Activation of Ratio-Peroxyfluor-1
RPF1)
(
,2
2 2
tion is far from benign, however, as overproduction of H O and
other reactive oxygen species (ROS) from the mitochondrial elec-
tron transport chain leads to oxidative stress and the subsequent
3
functional decline of organ systems. Accumulation of oxidative
damage over time is connected to debilitating human diseases where
age is a risk factor, including Alzheimer’s and related neurodegenera-
4
5
6
tive diseases, as well as cardiovascular disorders and cancer.
The far-ranging impacts of H homeostasis on human health
2
O
2
and disease provide motivation to devise new diagnostic methods
for detecting and quantifying its production from endogenous
sources. Synthetic fluorescent probes offer one approach to this
2 2
goal, and reagents that respond to H O by an emission increase
have been reported recently.7
-14
These intensity-based probes are
of practical value, but their application toward quantitative measure-
ments of changes in H concentrations in heterogeneous biologi-
2 2
O
cal samples can be complicated by variations in excitation intensity,
emission collection efficiency, sample thickness, and/or probe
concentration and environment. Ratiometric probes that afford
simultaneous recording of two measurable signals in the presence
and absence of analyte minimize these factors and can, in principle,
15
allow for accurate and quantitative readouts. To this end, we now
present the synthesis and properties of Ratio-Peroxyfluor-1 (RPF1),
a new ratiometric fluorescent reporter for H
selectivity for H over other ROS in water, visible wavelength
excitation and emission profiles, and is capable of detecting
endogenous H production from viable mitochondria.
Our strategy for ratiometric detection of H is based on
2 2
O . RPF1 features good
2
O
2
2 2
O
2 2
O
modulating fluorescence resonance energy transfer (FRET) in a two-
fluorophore cassette comprised of a coumarin donor and a boronate-
protected fluorescein acceptor linked by a rigid spacer. This
approach to controlling FRET by electronic spectral overlap as
opposed to modulating the physical separation of donor and acceptor
dyes is inspired by optical reporters of phosphatase activity.16 In
2 2
the absence of H O , the boronate protecting groups force the
acceptor to adopt a closed, colorless, and nonfluorescent lactone
form. Spectral overlap between coumarin emission and fluorescein
absorption is minimized, FRET is suppressed, and only blue donor
emission is observed upon excitation of the coumarin chromophore.
RPF1 was evaluated under simulated physiological conditions
DPBS buffer, 1% FBS, pH 7.4). As expected, the optical properties
(
of the parent cassette are dominated by the coumarin chromophore.
RPF1 displays a single absorption band in the visible region center-
ed at 420 nm, with blue-colored fluorescence from a corresponding
emission band centered at 464 nm (Figure 1a). The spectral data
are consistent with minimal FRET from the coumarin donor to the
closed, colorless fluoran acceptor. Upon treatment with H O , exci-
2 2
tation at 420 nm produces a bright green-colored fluorescence. The
resulting emission spectrum possesses one major band centered at
2 2
Upon selective reaction with H O to generate the open, colored,
and fluorescent fluorescein moiety, the acceptor shows a strong
absorption in the coumarin emission region. Spectral overlap is
enhanced, and excitation of the donor coumarin chromophore results
in increased green fluorescein acceptor emission by FRET. Changes
in [H O ] can be detected by measuring the ratio of blue and green
2 2
fluorescence intensities. Scheme 1 outlines the synthesis and
proposed activation of RPF1 based on this design.
517 nm with a minor band at 461 nm, consistent with increased
FRET from the coumarin donor to the open, colored fluorescein
acceptor (Figure 1a). The fluorescence response is accompanied by
concomitant growth of a visible wavelength absorption band
†
Department of Chemistry.
Department of Molecular and Cell Biology.
‡
9640
9
J. AM. CHEM. SOC. 2006, 128, 9640-9641
10.1021/ja063308k CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Our next goal was to evaluate the ability of RPF1 to detect
endogenous production of H O from living biological samples.
2 2
Assays employed purified mitochondria from Saccharomyces
cereVisiae. The yeast mitochondria were stimulated with antimycin
A (0.54 mg/mL), a cytochrome bc
inhibitor,18 to trigger generation
1
of H and other ROS by uncoupling of the respiratory electron
2
O
2
transport chain. Samples were treated with antimycin A for various
times and analyzed directly with RPF1. The ratiometric emission
data collected in Figure 1c show clear increases in H
from antimycin A-inhibited mitochondria over untreated control
samples; H levels detected by RPF1 (0.2 µM/min) are within
ranges reported using other analytical techniques.
2 2
O production
2
O
2
19,20
In addition,
control experiments show that RPF1 does not react with antimycin
A, and stimulated mitochondria without dye give no fluorescence.
The results demonstrate that RPF1 is capable of monitoring and
quantifying changes in endogenous [H
fluorescence response.
2 2
O ] through a ratiometric
To summarize, we have presented the synthesis and properties
of RPF1, a new type of ratiometric fluorescence reporter for
hydrogen peroxide. This FRET-based reagent features good selec-
2 2
tivity for H O over competing ROS as well as visible wavelength
excitation and emission profiles to minimize damage and autof-
luorescence from biological samples. Experiments with viable
mitochondria show that RPF1 can detect and quantify endogenous
H O production, establishing the potential utility of this approach
2 2
for probing peroxide biology in living systems.
Acknowledgment. We thank the University of California,
Berkeley, the Dreyfus Foundation, the Beckman Foundation, and
the American Federation for Aging Research for funding. A.E.A.
is supported by a Chemical Biology Interface Training Grant from
the NIH (T32 GM066698) and a Berkeley Chancellor’s Opportunity
Fellowship. We thank Prof. David Drubin for supplying mitochon-
dria, and Eric Paradise for help with artwork.
Figure 1. (a) Ratiometric fluorescence response of 1 µM RPF1 to 200
µM H2O2. Spectra shown were acquired before H2O2 addition and 5, 10,
1
5, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min after H2O2 was added. (b)
Relative reactivities of 1 µM RPF1 to various ROS. (1) H2O2; (2) tert-
-
+
t
butyl hydroperoxide (TBHP); (3) O2 ; (4) NO; (5) NO ; (6) •OH; (7) •O -
-
1
-
Bu; (8) OCl ; (9) O3; (10) O2. Data shown are for 10 mM O2 , 2 mM
O2, and 200 µM for all other ROS. Bars represent emission intensity ratios
1
Supporting Information Available: Synthetic and experimental
details (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
at 5 (white), 15 (light gray), 30 (gray), 45 (dark gray), and 60 min (black)
after addition of the appropriate ROS. (c) Fluorometric analysis of H2O2
produced by viable yeast mitochondria using 1 µM RPF1. Bars show
emission intensity ratios for untreated control mitochondria (white) and
mitochondria stimulated with the cytochrome bc1 inhibitor antimycin A
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J. AM. CHEM. SOC.
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VOL. 128, NO. 30, 2006 9641