Quantification of singlet oxygen production in the reaction of superoxide with hydrogen peroxide using a selective chemiluminescent probe

Article abstract of DOI:10.1021/ja052045b

A sensitive chemiluminescent probe that selectively reacts with singlet oxygen in the presence of superoxide and hydrogen peroxide has been used to quantify the production of singlet oxygen in the reaction of superoxide with hydrogen peroxide. The yield o

Full text of DOI:10.1021/ja052045b

Published on Web 06/04/2005
Quantification of Singlet Oxygen Production in the Reaction of Superoxide
with Hydrogen Peroxide Using a Selective Chemiluminescent Probe
Laura A. MacManus-Spencer and Kristopher McNeill*
Department of Chemistry, UniVersity of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455
Received March 31, 2005; E-mail: mcneill@chem.umn.edu
Superoxide radical anion (O
2
-•)1 and hydrogen peroxide (H
O
2 2
)2
Scheme 1
are formed intracellularly, and the reaction of these two reactive
oxygen species (ROS) has been studied for decades in an effort to
3,4
explain their observed “toxic synergism”. Both singlet oxygen
1
•
(
2
O ) and hydroxyl radical ( OH) have been proposed as highly
cytotoxic products of this reaction. Singlet oxygen is known to
5
oxidize a variety of biological substrates, such as proteins, certain
6
7
amino acids, and nucleic acids. Hydroxyl radical is a nonspecific
oxidant, reacting with proteins and free amino acids with rate
7
10
-1 -1 8
constants ranging from 10 to 10
The formation of both O
M
s .
was followed by analyzing aliquots of the reaction mixture over a
period of 90 min (Figure 1, circles). The formation of dioxetane 2
followed apparent first-order kinetics with an observed rate constant,
1
•
-•
2
and OH in the reaction of O
2
with
2 2
H O has been proposed to occur via the so-called Haber-Weiss
9
or Haber-Willst a¨ tter reaction (eq 1), in which a fraction of the
-3 -1
1
k
obs, of (2.1 ( 0.3) × 10
2
s . The yield of O under these
1
O
2
is produced as O
2
. The biological relevance of this reaction
-6
conditions was calculated to be (4.0 ( 0.4) × 10 M, which
4,10-12
has been debated for at least four decades.
-•
corresponds to a yield of (0.20 ( 0.03)% relative to the initial O
2
concentration.
The formation of O
-•
•
-
1
H O + O f O + OH + OH
(1)
2
2
2
2
2
in this system was supported by experi-
ments in which its lifetime was increased through the use of
1
1
In 1975, Kellogg and Fridovich suggested that O
2
could be
2
deuterated solvent and decreased by the addition of a O quencher.
1
formed in the Haber-Weiss reaction and provided experimental
evidence of its production in the reaction of xanthine oxidase with
acetaldehyde in an aqueous system, which simultaneously generates
production in the
Haber-Weiss reaction has also been found in aprotic solvents,
which are useful models of the nonaqueous hydrophobic environ-
ment of lipid bilayers.15
In this study, we used a sensitive chemiluminescent probe that
2
The possible reaction and deactivation pathways of O in this
-
•
system are illustrated in Scheme 2. When the reaction of O
was carried out in 57% toluene-d (Figure 1, squares), the
2
observed enhancement in the O yield (1.7) closely matched the
2
with
H O
2 2
8
-
•
13
1
1
O
2
and H
2
O .
2
Experimental evidence of O
2
14
1
enhancement predicted from the O
2
lifetimes in the two solvent
1
mixtures (2.0). The formation of O was also predictably inhibited
2
by the addition of 1,4-diazabicyclo[2.2.2]octane (DABCO), a known
1
21
O
2
quencher (Figure 1, triangles).
It has been proposed that the Haber-Weiss reaction (eq 1) does
1
-•
selectively reacts with O
2
in the presence of O
2
and H
2 2
O to
1
4,11
quantify the production of O
The trap-and-trigger probe, which we have described previously,
is based on a stable dioxetane precursor (1, Scheme 1). This
2
in the reaction of these two ROS.
not occur in the absence of a metal catalyst.
We found no
16
evidence for the participation of trace metal impurities in the
1
formation of O
2
from KO
2
and H
2
O
2
, as there were no differences
1
7
18
1
detection method builds upon the work of Schaap, Adam, and
others,19 who have reported a series of spiroadamantylidene-
substituted dioxetanes that are unusually stable and require a
in the apparent kinetics or O
2
yield in the presence and in the
absence of diethylenetriaminepentaacetic acid (DTPA), a metal
chelator. However, in the presence of a relatively high amount of
1
chemical trigger to initiate their chemiluminescent decomposition.
added Fe(II) acetate (100 µM), a 6-fold increase in the O
2
yield
1
In this detection scheme, O
2
is trapped in the form of a stable
and an almost 2-fold increase in the apparent first-order rate constant
dioxetane (2, Scheme 1), which is quantified by its chemilumin-
escence (CL) signal, triggered by the addition of tetra-n-butylam-
monium fluoride (TBAF).
were observed (see Supporting Information), suggesting that the
-
•
1
reaction of O
2
2 2 2
with H O to produce O can be catalyzed by
Fe(II) and possibly other redox-active metals. It is unclear from
-•
1
The reaction of O
2
with H O
2 2
was carried out in aprotic solvent
2
these experiments how the Fe(II) facilitates the production of O ,
(
toluene) to take advantage of the greater stability and higher
although it has been suggested that it can catalyze the reaction by
-
•
20
4,9
reactivity of O
2
under such conditions. To circumvent the
acting as a redox mediator.
The potential formation of OH in the reaction of O
•
-•
complications associated with heterogeneous reaction conditions
or the presence of water, a homogeneous solution of H
0.032 M) was prepared in toluene by the oxidation of 2-ethylanthra-
2
2 2
with H O
2
O
2
under the conditions used in this study was also investigated.
Hydroxyl radical is known to react with toluene with a rate constant
(
9
-1 -1
hydroquinone (2-EAHQ) and purified by distillation (see Supporting
Information).
of 8.1 × 10 M
s
to preferentially form o-, m-, and p-cresols
23
(as opposed to benzyl radical); the relative yields of these products
24
The formation of dioxetane 2 in the reaction of KO
2
(2 mM,
are reported to be 0.84, 0.41, and 1.0, respectively. A spectro-
photometric detection method using Gibbs’s reagent25 (2,6-dichloro-
quinone-4-chloroimide) was used to quantify the total cresol
solubilized with 18-crown-6 ether; see Supporting Information for
the determination of the dissolved O
-
•
2
2 2
concentration) with H O
•
(10 mM) in toluene at 25 °C in the presence of 100 µM probe 1
concentration as an indicator of OH production. By this method,
8954
9
J. AM. CHEM. SOC. 2005, 127, 8954-8955
10.1021/ja052045b CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. Support has been provided by a Grant-in-
Aid from the University of Minnesota.
Supporting Information Available: Experimental details for the
preparation of 2-EAHQ and the generation of H
2
O
2
in toluene; the
-•
reactions of O
2
with H O , 2-nitrobenzoic acid, and tert-butyl alcohol;
2 2
and the detection of cresols using Gibbs’s method. This material is
available free of charge via the Internet at http://pubs.acs.org.
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28
1
a
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(
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JA052045B
J. AM. CHEM. SOC.
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VOL. 127, NO. 25, 2005 8955