THE JOURNAL OF BIOLOGICAL CHEMISTRY

2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 27, Issue of July 6, pp. 2462124626, 2001

Printed in U.S.A.

The Carbonate Radical Is a Site-selective Oxidizing Agent of

Guanine in Double-stranded Oligonucleotides*
Received for publication, February 6, 2001, and in revised form, April 24, 2001
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.M101131200

Vladimir Shafirovich, Alexander Dourandin, Weidong Huang, and Nicholas E. Geacintov

From the Chemistry Department and Radiation and Solid State Laboratory, New York University,
New York, New York 10003-5180

There is growing evidence that bicarbonate and carbon dioxide, both present in biological systems in significant amounts,
can alter the mechanisms and reaction pathways of reactive
oxygen (1 4) and nitrogen (513) species formed during normal
metabolic activity and under conditions of oxidative stress. It
has been proposed that the mechanism of generation of carbonate radical anions (CO3. )1 from bicarbonate (HCO3) or CO2 can
* This work was supported by National Science Foundation Grant
CHE-9700429 and by a grant from the Kresge Foundation. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Chemistry Dept. and
Radiation and Solid State Laboratory, 31 Washington Place, New York
University, New York, NY 10003-5180. Tel.: 212-998-8456; Fax: 212998-8421; E-mail: vs5@nyu.edu.
1
The abbreviations used are: CO3. , carbonate radical anion; HCO3,
bicarbonate anion; 8-oxo-dG, 8-oxo-7,8-dihydro-2-deoxyguanosine;
This paper is available on line at http://www.jbc.org

involve the one-electron oxidation of HCO3 at the active site of

copper-zinc superoxide dismutase (3, 4) and homolysis of the
nitrosoperoxycarbonate anion (ONOOCO2) formed by the reaction of peroxynitrite with carbon dioxide (14 18).
The carbonate radical anion is a strong one-electron oxidant
that oxidizes appropriate electron donors via electron transfer
mechanisms (19). Detailed pulse radiolysis studies have shown
that carbonate radicals can rapidly abstract electrons from
aromatic amino acids (tyrosine and tryptophan). However, reactions of CO3. with sulfur-containing methionine and cysteine
are less efficient (20 22). Hydrogen atom abstraction by carbonate radicals is generally very slow (19), and their reactivities with other amino acids are negligible (20 22). It is well
established that carbonate radicals can play an important role
in the modification of selective amino acids in proteins in cellular environments under conditions of oxidative stress, aging,
and inflammatory processes (1, 11, 12).
The role of HCO3/CO2 in potentiating oxidative DNA damage has received relatively little attention. It has been shown
that the presence of HCO3/CO2 inhibits direct strand cleavage
of DNA induced by ONOO but enhances the formation of
8-nitroguanine, alkali-labile and formamidopyrimidine glycosylase-labile DNA lesions (2325). Peroxynitrite causes direct
DNA strand cleavage by oxidizing deoxyribose. However, in the
presence of HCO3/CO2 there is a shift in product distribution
from direct strand cleavage to the formation of oxidative modifications of guanines (26), suggesting that the carbonate radical anion could play an important role in this phenomenon
(24). Although guanine is indeed the most easily oxidized base
in DNA, the reactions of the carbonate radical anions with the
different aromatic DNA residues have not yet been
characterized.
In this work, we explore the electron transfer reactions from
guanine electron donor residues embedded in the self-complementary hexadecanucleotide duplex d(AACGCGAATTCGCGTT) to CO 3. electron acceptor. Employing transient
absorbance laser flash photolysis techniques, we monitored
simultaneously the kinetics of disappearance of the CO3. anion
radical and the appearance of guanine radicals by transient
absorption spectroscopy techniques. The decay of the CO3. radical anion was followed by monitoring its absorbance (max
600 nm), whereas the concomitant formation of guanine neutral radicals, G(H), was identified by their characteristic
narrow absorption band at 315 nm. These observations constitute the first direct indication that carbonate radicals are site-selective one-electron oxidants of guanine residues in doubleONOOCO2, nitrosoperoxycarbonate anion; ONOO, peroxynitrite;
SO4. sulfate radical; S2O82, persulfate; dG, 2-deoxyguanosine; dGMP,
2-deoxyguanosine 5-monophosphate; NO, nitric oxide; G(H), guanine neutral radical; NHE, normal hydrogen electrode; 2AP,
2-aminopurine.

24621

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The carbonate radical anion (CO3. ) is believed to be an

important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO2 with ONOO)
in cellular environments. Employing nanosecond laser
flash photolysis methods, we show that the CO3. anion
can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT)
dissolved in air-equilibrated aqueous buffer solution
(pH 7.5). In these time-resolved transient absorbance
experiments, the CO3. radicals are generated by oneelectron oxidation of the bicarbonate anions (HCO3)
with sulfate radical anions (SO4. ) that, in turn, are derived from the photodissociation of persulfate anions
(S2O82) initiated by 308-nm XeCl excimer laser pulse
excitation. The kinetics of the CO3. anion and neutral
guanine radicals, G(H), arising from the rapid deprotonation of the guanine radical cation, are monitored
via their transient absorption spectra (characteristic
maxima at 600 and 315 nm, respectively) on time scales
of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO3. is (1.9 0.2) 107 M1 s1. The decay of the
CO3. anions and the formation of G(H) radicals are
correlated with one another on the millisecond time
scale, whereas the neutral guanine radicals decay on
time scales of seconds. Alkali-labile guanine lesions are
produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The
DNA fragments thus formed are identified by a standard
polyacrylamide gel electrophoresis assay, showing that
strand cleavage occurs at the guanine sites only. The
biological implications of these oxidative processes are
discussed.

24622

Carbonate Radical Is a Site-selective Oxidizing Agent

TABLE I
Oxidation of the d(AACG1CG2AATTCG3CG4TT) duplex by CO3. radicals in aqueous buffer solutions (pH 7.5)
Reaction schemes and rate constants are shown.
No.

Reaction

k
M

Reference
1

S2O82 h 3 2 SO4.
SO4. SO4. 3 S2O82

stranded DNA and do not react significantly with any of the

other nucleic acid bases A, C, or T. The oxidation of the
different guanines in the self-complementary DNA duplex
results in alkali-labile guanine lesions; these are revealed by
the site-selective DNA strand cleavage induced by a standard
hot piperidine treatment and polyacrylamide gel electrophoresis assay.
EXPERIMENTAL PROCEDURES

Materials2-Deoxyguanosine 5-monophosphate (dGMP), 8-oxo7,8-dihydro-2-deoxyguanosine (8-oxo-dG), and all inorganic salts

(99% purity) were obtained from Sigma-Aldrich and were used as
received. The d(AACGCGAATTCGCGTT) oligonucleotide was synthesized by standard automated phosphoramidite chemistry techniques.
The tritylated oligonucleotide was removed from the solid support and
deprotected with concentrated ammonium hydroxide. The crude oligonucleotide was purified by reversed phase high performance liquid
chromatography and detritylated in 80% acetic acid according to standard protocols.
The oligonucleotide was dissolved in 20 mM phosphate buffer (pH 7.5)
containing 100 mM Na2SO4. Annealing of the two strands was accomplished by heating the samples to 90 C for 10 min and then allowing
the samples to cool slowly back to room temperature overnight.
Laser Flash PhotolysisThe transient absorption spectra were recorded using a pulsed laser excitation source and a kinetic spectrometer
system (response time, 7 ns) described earlier (2730). Briefly, this
system consists of a pulsed Lambda Physik EMG 160 MSC XeCl excimer laser (308 nm; full width at half-maximum pulse width, 12 ns;
energy, 70 mJ pulse1 cm2; 0.1 Hz), and a 75 W pulsed xenon lamp
to probe the transient absorption kinetics. The probe flash was passed
through a McPherson monochromator and was monitored by a
Hamamatsu R928 phomultiplier; its output was recorded and digitized
using a Tektronix TDS 620 oscilloscope. All experiments, including data
collection and analysis, were controlled by a computer.
The CO3. radical anions were generated by oxidation of HCO3 with
SO4. radical anions (31, 32). The SO4. ions were generated by the photodissociation of persulfate anions (31, 33). Although persulfate ions
exhibit negligible reactivity with nucleosides and oligonucleotides, the
SO4. radical anions are known to react with the DNA bases. The triggered dissociation of persulfate ions into SO4. radical anions in timeresolved pulse radiolysis or flash photolysis experiments has been previously employed by Steenken and co-workers (34 36), ONeill and
Davies (37), and Bachler and Hildenbrand (38), to study the kinetics of
reactions of the SO4. radical anions with nucleic acids.
Air-equilibrated buffer solutions containing 20 100 M oligonucleotide duplexes, 300 mM NaHCO3, and 25 mM Na2S2O8 were placed in a
0.5-ml quartz flow cell (80% transmittance at 200 nm) from NSG
Precision Cells, Inc. The optical pathlength of this cell was 1 cm, and the
cell was aligned parallel to the direction of the xenon lamp probe light
beam. After one laser shot, the irradiated solution was replaced by a
fresh sample solution employing a computer-controlled flow system. All

laser flash photolysis experiments were performed at 20 C.

Pseudo-first order rate constants (kn) of the radical (R) substrate
(S) reactions and the second order rate constants of the radical recombination were determined by least squares fits of the appropriate,
indicated kinetic equations to the transient absorption profiles obtained
in five different experiments with five different samples. The second
order rate constants of the R S reactions were calculated by a least
square best fit to a linear dependence of the kn versus [S] plots.
Steady-state SpectroscopyRoutine UV absorption spectra and UV
melting profiles were measured using an HP 84453 diode array spectrophotometer with an HP 89090A Peltier temperature control unit
(Hewlett Packard GMBH, Waldbronn, Germany).
Oxidative DNA Strand Cleavage AssayThe oligonucleotides were
labeled at their 5 termini by using T4 polynucleotide kinase (Amersham Pharmacia Biotech) and [-32P]ATP (PerkinElmer Life Sciences)
as described previously (27). The 10-l samples containing 32P 5-endlabeled oligonucleotide strands in 2 2-mm square pyrex capillary
tubes (Vitrocom, Inc.) were irradiated with 308-nm laser light. After the
irradiation, the reaction mixture was quenched by the addition of
-mercaptoethanol and was heated for 30 min at 90 C in 100 l of a 1
M piperidine solution. The samples were vacuum dried and assayed by
a denaturing polyacrylamide gel electrophoresis (20% acrylamide, 19:1
bis ratio, 7 M urea), as described previously (27). The relative intensities
of cleavage were analyzed using a Bio-Rad 250 imaging system.
RESULTS

Duplex DesignThe duplex formed by the self-complementary 16-mer d(AACG1CG2AATTCG3CG4TT) sequence contains
the 12-mer d(CG1CG2AATTCG3CG4) core. The latter is the
first oligonucleotide duplex containing at least one turn of helix
for which structural information was obtained by single-crystal
x-ray diffraction methods (39, 40) and high resolution NMR
spectroscopy (41, 42). We added one AA and one TT doublet to
the two ends of this 12-mer to diminish the known fraying (42)
of the G:C base pairs at or near the ends of the oligonucleotide.
The 16-mer duplex exhibits well defined cooperative melting
curves. The average melting temperature (Tm 67 1 C) and
the hyperchromicity of 17% were calculated from plots of the
absorbance (260 nm) versus temperature obtained from heating-cooling cycles (43).
Generation of CO3. Radicals by the Oxidation of HCO3 by SO4.
RadicalsThe kinetic parameters associated with the reactions
of HCO3 and S2O82 in aqueous solutions induced by laser pulse
excitation are well documented (19). The excitation of S2O82 in
aqueous solutions with 308-nm XeCl excimer laser pulses generates the SO4. radical anions (Table I, Reaction 1) with a quantum
yield of 0.55 (33). The SO4. radicals exhibit transient absorption
bands at 445 nm with an extinction coefficients of 1600 M1 cm1

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308 0.55
Ref. 33
k2 (1.1 0.1) 109
This work
k2 1.35 109
Ref. 33
3
SO4. HCO3 3 SO42 CO3.
k3 (4.6 0.5) 106
This work
6
k3 9.1 10
Ref. 31
6
k3 2.8 10
Ref. 32
.
.
2
7
4
CO3 CO3 3 CO4 CO2
k4 (1.3 0.1) 10
This work
7
k4 2 10
Ref. 45
5a
CO3. d[G-oligo] 3 CO32 d[G(H)oligo]
k5 (1.9 0.2) 107
This work
6a
CO3. dGMP 3 CO32 dGMP(H)
k6 (6.6 0.7) 107
This work
7b
CO3. dAMP 3
k7 2.8 106
This work
8b
CO3. dCMP 3
k8 1.9 106
This work
.
a
6
9
CO3 dTMP 3
k9 1.7 10
This work
.
2
a
8

10
CO3 8-oxo-dG 3 CO3 8-oxo-G(H)
k10 (7.9 0.8) 10
This work
.
2
b
9
11
SO4 d[G-oligo] 3 SO4 d[G(H)oligo]
k11 (9.5 0.9) 10
This work
a
The reactions were monitored spectroscopically via the decay of CO3. (600 nm) and SO4. (445 nm) radicals and the concomitant formation of the
product radicals, G(H) (315 nm) and 8-oxo-G(H) (320 nm).
b
The reactions were monitored by the accelerated decay of CO3. (600 nm) and SO4. (445 nm) radicals in the presence of the indicated substrate
molecules.
1
2

Carbonate Radical Is a Site-selective Oxidizing Agent

(44). In pure aqueous buffer solutions, the SO4. radical anions

decay mostly via bimolecular recombination processes (Table I,
Reaction 2). In the presence of HCO3, the decay of SO4. monitored
at 445 nm results in the formation of the CO3. radicals, identified
by their characteristic absorption band with a maximum at 600
nm and molar extinction coefficient of 1970 M1 cm1 (45). This
effect is attributed to the reduction of SO4. by HCO3 with the
formation of CO3. (Table I, Reaction 3); the HCO3. radical, the
primary product of the one-electron oxidation of HCO3, is a very
strong acid (pKa 0) and thus deprotonates rapidly in aqueous
solutions (46). The recombination of the CO3. radicals is a slow
process (Table I, Reaction 4) occurring via transfer of O . anions
from one CO3. radical to another, thus forming carbon dioxide and
peroxymonocarbonate (47). The rate constants of reactions 2 4
measured during the course of this work are also summarized in
Table I; these values differ only slightly from published values
because of differences in ionic strength (45).
Transient Absorption Studies of the Oxidation of Guanine by
CO3. Radicals in DNA DuplexesTypical transient absorption
spectra of a solution of d(AACGCGAATTCGCGTT) duplexes (50
M) containing 25 mM S2O82 and 300 mM HCO3, recorded at
various delay times (t) after 308-nm laser pulse excitation, are
shown in Fig. 1. The transient absorption spectrum recorded at
t 60 s exhibiting a maximum at 600 nm corresponds to the
well known spectrum of the CO3. radical (31, 45, 46); at this
HCO3 concentration (300 mM), the decay of the SO4. anion radicals occurs rapidly (within 0.7 s) and is not shown in Fig. 1.
The decay of the CO3. absorption band is accompanied by the rise
of another, narrow transient absorption band centered at 315
nm. This spectrum, recorded at t 3.9 ms, is characteristic of
both the guanine radical cation, G, and the neutral guanine
radical, G(H) (34). Thus, the carbonate radical is a site-selective one-electron oxidant of guanine residues in double-stranded
DNA (Table I, Reaction 5). As shown below (DNA strand cleavage
assay), the oxidation of the other three DNA bases by CO3. anion
radicals is significantly less efficient than that of guanine. This is
consistent with the relative rate constants of one-electron oxidation of 2-deoxynucleotide monophosphates by CO3. radicals. In

the case of dGMP, the rate constant is some 20 40 times greater

than the rate constant determined for dAMP, dCMP, and dTMP
(Table I, Reactions 6 9). However, the rate constant of oxidation
of 8-oxo-dG is greater by a factor of 10 (Table I, Reaction 10)
than that of dGMP (36).
Deprotonation of Guanine Radical CationsThe pKa of free
guanine radical cations, G, generated by the one-electron
oxidation of dG or dGMP, is 3.9 (34). Therefore, in neutral
aqueous solutions, deprotonation of G occurs rapidly (rate
constant k 2 106 s1). Candeias and Steenken (34) proposed that in double-stranded DNA, with the G radical hydrogen-bonded to its partner Watson-Crick cytosine residue
(pKa 4.45), the deprotonation rate constant of G should be
even greater than in the case of free G. The neutral G(H)
radical has indeed been detected by electron spin resonance
techniques upon oxidation of double-stranded DNA in aqueous
solutions at room temperature (48, 49). Hence, the guanine
radicals observed on millisecond time scales (Fig. 1) are neutral
radicals, G(H), rather than G radical cations.
Kinetics of Guanine Oxidation by CO3. Radicals in DNA DuplexesIn the absence of DNA, the bimolecular recombination
of two CO3. radicals occurs, and the rate constant (k4) of this
bimolecular reaction has been reported (Table I, Reaction 4).
Consequently, the decay profile of CO3. radical, monitored at
600 nm (Fig. 1), can be described by mixed first and second
order kinetics.
d[CO3. ]/dt k5CO3. ] 2k4CO3. ]2

(Eq. 1)

The pseudo-first order rate constant k5 k5 [DNA], where

[DNA] is the concentration of DNA duplexes, and k5 is the rate
constant of reaction of the CO3. anion radicals with the DNA
duplexes. Defining the initial concentration of carbonate radical anions (at t 0) as [CO3. ]0, the solution of Equation 1 yields
the following expression for the time dependence of the CO .

radical concentration.
CO3. t k5CO3. 0/k5 2k4CO3. ]0)expk5t 2k4CO3. ]0}

(Eq. 2)

The value of k5 was determined by fitting Equation 2 to the

CO3. decay profiles measured at 600 nm and at different DNA
concentrations. The value of k4 was determined in the absence
of the DNA duplex (Table I). As expected, k5 increases linearly
with the concentration of the DNA duplex (Fig. 2), yielding a
value of k5 1.9 107 M1 s1. The rate of oxidation of dGMP
by CO3. radicals was determined by similar experiments (data
not shown), and the rate constant k6 6.6 107 M1 s1 (Table
I). The rate constant of oxidation of free dGMP (k6) is 3.5
times larger than k5, the value of the rate constant for the
oxidation of G within the DNA duplex. The apparent decrease
in the reactivity of guanines in the duplexes is probably associated with a decreased accessibility of guanines to the CO3.
radicals (see below).
Decay Kinetics of G(H) RadicalsThe decay of the G(H)
radicals in the double-stranded oligonucleotide is shown on two
different time scales in Fig. 3. On the 100-ms time scale (Fig.
3, inset), the change in G(H) radical concentrations is too
small to be measurable, but a slow decay is evident on a time
scale of 0.8 s (Fig. 3).
Chemical Damage in Double-stranded DNA Induced by CO3.
RadicalsThe rate constant of reaction of SO4. radicals with
DNA is greater than that of CO3. radicals (Table I, Reactions 11
and 5, respectively). The value of k11 5 109 M1 s1 (Table
I) was determined from the kinetic decay profiles of the SO .
4

radical anions monitored at 445 nm (absorption maximum of

SO4. ) in solution containing a 50 M concentration of the
d(AACGCGAATTCGCGTT) duplex and 25 mM S2O82. This

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FIG. 1. Kinetics of oxidation of guanine in the duplex

d(AACGCGAATTCGCGTT) (50 M) by CO3. radicals in air-equilibrated buffer solution containing 25 mM S2O82 and 300 mM
HCO3 ions (pH 7.5). The CO3. radicals were indirectly generated (see
text) by SO4. radicals obtained by the photolysis of S2O82 with 308 nm
excimer laser pulses (70 mJ/pulse/cm2). The transient absorption
spectra were recorded at various delay times (t) after the excitation.
The 600 nm maximum is due to CO3. radical anions, and the 315 nm
maximum is due to G(H) radicals. The lines are shown for visualization only.

24623

24624

Carbonate Radical Is a Site-selective Oxidizing Agent

FIG. 3. Decay kinetics of the 315 nm absorbance of the guanine

radicals, G(H), in the self-complementary duplex d(AAC GCGAATTCGCGTT) (100 M) in 25 mM S2O82 and 300 mM HCO3
buffer solution, following excitation with a 308 nm excimer
laser pulse (70 mJ/pulse/cm2).

constant is close to the rate constant of the oxidation of dG by

SO4. radicals (k 2.3 109 M1 s1) reported by ONeill and
Davies (37). To minimize the generation of G(H) by SO4.
radicals in the DNA cleavage experiments and to focus on the
reaction of CO3. radicals with the DNA duplex, a 300 mM concentration of HCO3 was selected in these experiments. Under
these conditions, the SO4. radicals decay predominately via
reactions with HCO3, and their lifetimes are less than 0.7 s
(data not shown). Thus, reactions of CO3. with the DNA duplex
are dominant.
DNA Strand CleavageThese experiments were performed
with the aim of determining whether all guanines are uniformly damaged after reaction with CO3. radical anions. Oxidative guanine base damage in DNA can be revealed by treatment with hot piperidine solutions, which gives rise to strand

FIG. 4. Gel electrophoresis patterns of irradiated, hot-piperidine oligonucleotide strands. Shown is an autoradiograph of a denaturating gel (7 M urea, 20% polyacrylamide gel) showing the cleavage
patterns induced by 308-nm excimer laser pulse excitation (20 mJ/
pulse/cm2, 10 Hz) of the 32P 5-end-labeled d(AACG1CG2AATTCG3CG4TT) strands in the duplex form (10 M) in air-equilibrated buffer
solutions (pH 7.5) containing 10 mM S2O82 and 300 mM HCO3. After the
irradiation, the oligonucleotide solutions were treated with hot piperidine (1 M, 90 C) for 30 min and loaded onto a polyacrylamide gel. A,
lane G, Maxam-Gilbert G sequencing reaction; lane 1, unirradiated
duplex (without piperidine treatment); lane 2, unirradiated duplex (after hot piperidine treatment); lanes 3 and 5, irradiated duplex (without
piperidine treatment; integrated dosage received by the sample, 0.5 and
4 J/cm2, respectively); lanes 4 and 6, irradiated duplex (after hot piperidine treatment; integrated dosage received by the sample, 0.5 and 4
J/cm2, respectively). B, histogram obtained by scanning the autoradiogram (lane 4, 0.5 J/cm2); hot piperidine cleavage patterns at the different guanine sites G 1 , G 2 , G 3 , and G 4 in the irradiated, selfcomplementary 32P 5-end-labeled d(AACG1CG2AATTCG3CG4TT)
duplex. C, total fraction of strands cleaved at the dosage of 4 J/cm2, and
the fraction of fragments cleaved at all G, C, A, and T sites in the
self-complementary duplex (from lane 6). The error bars show the
standard deviations of the cleavage patterns (B and C) obtained in three
different photocleavage experiments.

cleavage at the damaged sites (50). The cleaved fragments thus

formed can be visualized by high resolution gel electrophoresis.
Typical results, obtained after irradiation and hot alkali treatment of a solution of a 10 M d(AACGCGAATTCGCGTT) duplex, 10 mM S2O82, and 300 mM HCO3 with 308-nm excimer
laser pulses, are depicted in the gel autoradiograph in Fig. 4A.
Oligonucleotide strand cleavage is negligible in the unirradiated control samples with or without hot piperidine treatment
(lanes 1 and 2, respectively). However, upon irradiation, cleavage is observed mostly at the guanine sites, and the extent of
strand cleavage is more pronounced after hot piperidine treatment (lanes 4 and 6) than before (lanes 3 and 5). In lanes 3 and
4 the total energy dosage received by the sample was 0.4 J/cm2,
whereas in lanes 4 and 6 the dosage was approximately ten
times higher. The extent of damage increases with the laser
energy dosage. It is evident that the sites of cleavage correlate
well with the cleavage pattern obtained by the Maxam-Gilbert
sequencing reaction (lane G), clearly indicating that strand
cleavage occurs predominantly at the four different guanines in
the self-complementary oligonucleotide duplex. The relative
cleavage efficiencies at the different guanines are shown in Fig.
4B. The probability of strand cleavage at these sites differs,
being higher at sites G1 and G2, which are closer to the ends of
the oligonucleotide, than at sites G2 and G3, which are positioned closer to the center of the duplex. In these experiments

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FIG. 2. Dependence of the rate constant, k5, characterizing

the decay of CO3. radical anions, on the concentration of the
self-complementary d(AACGCGAATTCGCGTT) duplex in airequilibrated buffer solutions (pH 7.5) containing 25 mM S2O82
and 300 mM HCO3. The solid line represents a least squares, best
linear fit to the k5 data points, each of which represents an average of
five individual laser flash photolysis experiments.

Carbonate Radical Is a Site-selective Oxidizing Agent

the overall percentage of DNA strands cleaved was less than
5%, indicating that the DNA duplexes suffered, on average, less
than one DNA strand cleavage event/molecule (0.4 mJ/cm2
dosage). At the higher dosage, the fraction of cleaved DNA
strands increased to 40 45%. Nevertheless, strand cleavage at
sites other than guanine remains small (Fig. 4C). These results
clearly indicate that the CO3. radical anion is a site-selective
oxidizing agent of guanines in double-stranded DNA. These hot
piperidine-induced strand cleavage results are in good agreement with the spectroscopic laser photolysis data. The latter
results suggest that carbonate radical anions react by oneelectron transfer mechanisms more readily with guanine than
with the other three DNA bases.
DISCUSSION

ent base pair opening rates (42) and probably to steric hindrance
factors as well.
The Long Lifetimes of Guanine Radicals, G(H), in Doublestranded DNAIt is interesting to note that the lifetimes of the
G(H) radicals are significantly longer in the double-stranded
oligonucleotide (Fig. 3) than the lifetimes of either dGMP(H)
or dG(H) radicals in solution (27, 29, 57). Furthermore, the
lifetime of the G(H) radicals in the d(AACGCGAATTCGCGTT) duplex (Fig. 3) is orders of magnitudes longer than
in oligonucleotide duplexes in which these radicals are generated by one-electron transfer reactions from guanine to 2-aminopurine (2AP) radicals (28). In the latter case, as well as in the
present experiments, the lifetime measurements were conducted in air- or oxygen-equilibrated solutions. Thus, differences in the decay rates caused by reactions of G(H) with O2
can be ruled out. In the case of the 2AP modified duplexes, 2AP
radicals were generated by two-photon photoionization of 2AP
residues, thus generating hydrated electrons as well. The hydrated electrons are efficiently scavenged by molecular oxygen
resulting in the formation of O2. radicals (58). Hence, a possible
reason for the faster decay of the G(H) radicals in the 2APmodified duplexes is the fast reaction of G(H) radicals with
O2. radicals. Recently, Candeias and Steenken (59) have reported that the reaction of G(H) with O2. indeed occurs rapidly with a rate constant that is nearly diffusion-controlled
(3 109 M1 s1). In contrast, the reaction of G(H) with O2
(60) is very slow (k 102 M1 s1). A lifetime of G(H) in DNA
as long as 5 s has been reported (48). The presence or the
absence of O2. could thus be one crucial factor that determines
the lifetime of G(H) radicals in double-stranded DNA.
Multiple Pathways of CO3. Generation in Biological SystemsIt is generally accepted that aging, chronic inflammation, and diverse infectious disorders are associated with an
enhanced production of reactive oxygen species, like superoxide
radicals (O2. ), hydrogen peroxide (H2O2), and hypochlorous acid
(HOCl) (61 63). Hydroxyl radicals produced by the one-electron reduction of H2O2 in Fenton-type reactions are well known
species that can induce oxidative modifications and damage to
the DNA (50, 64). However, OH radicals are extremely reactive
and thus have a very limited range of diffusion (65). Therefore,
OH radicals induce damage to biomolecules only within the
immediate locale where they are generated. Wolcott et al. (66)
have proposed that OH radicals can oxidize HCO3 anions and
thus generate the CO3. radicals that are less reactive than the
OH radicals. Recently, Kalyanaraman and co-workers (3, 4)
have shown that the enhancement of peroxidase activity of
copper-zinc superoxide dismutase in the presence of HCO3 (67)
is associated with the generation of CO3. radicals by one-electron oxidation of HCO3 occurring at the enzyme active site.
Still another source of carbonate anion radicals might be of
importance. Inflammatory and infectious processes are associated with the generation of not only reactive oxygen species but
also of nitric oxide (NO) catalyzed by NO synthase type 2 (68,
69). Beckman and co-workers (70) have proposed that the stimulated generation of NO and O2. radicals in vivo can result in
the formation of the highly reactive and toxic ONOO. The
combination of NO and O2. occurs with a diffusion-controlled
rate constant, k (6.719) 109 M1 s1 (71, 72). That leads to
the formation of ONOO even at very low concentrations of
NO and O2. . Lymar and Hurst (6) have demonstrated that in
neutral aqueous solutions, ONOO rapidly reacts with CO2
(k 3 104 M1 s1) to yield highly unstable ONOOCO2.
Homolytic dissociation of ONOOCO2 occurring on submicrosecond time scales (73) produces CO3. and NO2 radicals with a
cage escape yield of 0.3 (16, 18).
Hence, because of the relatively high concentrations of

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Selective Oxidation of Guanines in DNA by CO3. Radicals

The selective reactivity of the CO3. radicals with guanine rather
than with any of the other three DNA bases, is a consequence
of the thermodynamic and kinetic characteristics of these electron donor/acceptor reactions. The redox potential of the CO3.
radicals at pH 7, estimated from the redox potential E0
(CO3. /CO32) 1.59 V versus NHE (51) and the pKa values of
6.37 (CO2, H2O/HCO3) and 10.25 (HCO3/CO32) (52), and pKa
0 (HCO3. /CO3. ) (46), is 1.7 V versus NHE. The selective
oxidation of guanines by CO3. radicals is thus thermodynamically feasible. Because the redox potential of free dG,
E7[dG(H)/dG] 1.29 V versus NHE is smaller than the redox
potential of dA, E7[dA(H)/dA] 1.42 V versus NHE and that
of the pyrimidine bases, E7 1.7 V (dT) and 1.6 V (dC) versus
NHE (35), it is reasonable that only guanine residues in DNA
are oxidized by CO3. radical anions. The oxidized guanine derivative 8-oxo-dG, a product of two-electron oxidation of guanine (50), is further oxidized by CO3. radicals with a greater
rate constant than in the case of guanine (Table I). This enhanced reactivity of 8-oxo-dG with CO3. radicals is correlated
with its lower redox potential, E7 0.74 V versus NHE (36),
which is smaller than the redox potentials of the other nucleosides discussed.
Rate Constants of Interaction of the CO3. Radicals with
GuaninesIt is interesting to note that the rate constant of
oxidation of dGMP by CO3. radical anions (k6 6.6 107 M1
s1; Table I) is much lower than the rate constants of dGMP
oxidation by aromatic radical cations with similar redox potentials. For instance, radical cations of the pyrene derivative
7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (E0 1.5 V
versus NHE; Ref. 53) and thioanisole (E0 1.45 V versus
NHE; Ref. 54) oxidize dGMP or dG with rate constants of
1.7 109 M1 s1 (55), and 1.4 109 M1 s1 (35), respectively. These results indicate that the reactivity of CO3. radicals with guanines is lower than expected from differences in
redox potentials alone. Schindler et al. (56) have shown that the
CO3. /CO32 system is characterized by a small self-exchange rate
constant (k 0.4 M1 s1) and a high internal reorganization
energy that results in a low reactivity of CO3. in bimolecular outer
sphere electron transfer reactions. Furthermore, in doublestranded DNA the rate constant of oxidation of guanines is even
lower than in the case of free dGMP. This value, k6 1.9 107
1 1
M
s (Table I), is averaged over the four different guanine sites
G1, G2, G3, and G4. The cleavage efficiencies (Fig. 4B) are lower
at the two central guanines G2 and G3 than at the outer guanines
G1 and G4. These variations in reactivities correlate roughly with
the imino proton exchange rates and thus the base pair opening
rates, in the different G:C base pairs in the 12-mer
d(CG1CG2AATTCG3CG4) studied by Patel and co-workers (42).
Thus, the lower reactivities of the CO3. radical anion with guanines in double-stranded DNA are most likely due to site-depend-

24625

24626

Carbonate Radical Is a Site-selective Oxidizing Agent

HCO3/CO2 in intracellular and interstitial fluids of up to 30

mM (74), response to aging, chronic inflammation, and diverse
infectious disorders might also include the enhanced generation of CO3. radicals that can damage not only proteins (1, 12)
but also DNA as shown in this report.
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The Carbonate Radical Is a Site-selective Oxidizing Agent of Guanine in

Double-stranded Oligonucleotides
Vladimir Shafirovich, Alexander Dourandin, Weidong Huang and Nicholas E. Geacintov
J. Biol. Chem. 2001, 276:24621-24626.
doi: 10.1074/jbc.M101131200 originally published online April 24, 2001

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