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INTRODUCTION |
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 (5-13) 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
(CO
3)1 from
bicarbonate (HCO
) or CO2
can involve the one-electron oxidation of
HCO
at the active site of copper-zinc
superoxide dismutase (3, 4) and homolysis of the nitrosoperoxycarbonate
anion (ONOOCO
) 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 CO
3 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 HCO
/CO2 in
potentiating oxidative DNA damage has received relatively little
attention. It has been shown that the presence of
HCO
/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 (23-25). Peroxynitrite causes direct
DNA strand cleavage by oxidizing deoxyribose. However, in the presence
of HCO
/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 CO
3 anion radical and the appearance of guanine radicals by transient absorption spectroscopy techniques. The decay of the CO
3 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 double-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.
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EXPERIMENTAL PROCEDURES |
Materials--
2'-Deoxyguanosine 5'-monophosphate (dGMP),
8-oxo-7,8-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 Photolysis--
The transient absorption spectra
were recorded using a pulsed laser excitation source and a kinetic
spectrometer system (response time, ~7 ns) described earlier
(27-30). 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 pulse
1
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 CO
3 radical anions were generated by oxidation of
HCO
with SO
4 radical anions
(31, 32). The SO
4 ions were generated by the photodissociation
of persulfate anions (31, 33). Although persulfate ions exhibit
negligible reactivity with nucleosides and oligonucleotides, the
SO
4 radical anions are known to react with the DNA bases. The
triggered dissociation of persulfate ions into SO
4 radical
anions in time-resolved pulse radiolysis or flash photolysis
experiments has been previously employed by Steenken and co-workers
(34-36), O'Neill and Davies (37), and Bachler and Hildenbrand (38),
to study the kinetics of reactions of the SO
4 radical anions
with nucleic acids.
Air-equilibrated buffer solutions containing 20-100 µM
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 Spectroscopy--
Routine 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 Assay--
The 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-µl
samples containing 32P 5'-end-labeled 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 µl 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 Design--
The 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 CO
3 Radicals by the Oxidation of
HCO
by SO
4
Radicals--
The kinetic parameters associated with the reactions of
HCO
and
S2O
in aqueous solutions
induced by laser pulse excitation are well documented (19). The
excitation of S2O
in
aqueous solutions with 308-nm XeCl excimer laser pulses generates the
SO
4 radical anions (Table I,
Reaction 1) with a quantum yield of 0.55 (33). The SO
4
radicals exhibit transient absorption bands at 445 nm with an
extinction coefficients of 1600 M
1
cm
1 (44). In pure aqueous buffer solutions, the
SO
4 radical anions decay mostly via bimolecular recombination
processes (Table I, Reaction 2). In the presence of
HCO
, the decay of SO
4
monitored at 445 nm results in the formation of the CO
3
radicals, identified by their characteristic absorption band with a
maximum at 600 nm and molar extinction coefficient of 1970 M
1 cm
1 (45). This effect is
attributed to the reduction of SO
4 by HCO
with the formation of
CO
3 (Table I, Reaction 3); the HCO
3 radical, the
primary product of the one-electron oxidation of
HCO
, is a very strong acid
(pKa < 0) and thus deprotonates rapidly in aqueous
solutions (46). The recombination of the CO
3 radicals is a
slow process (Table I, Reaction 4) occurring via transfer of
O
anions from one CO
3 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).
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Table I
Oxidation of the
d(AACG1CG2AATTCG3CG4TT)
duplex by CO 3 radicals in aqueous buffer solutions (pH 7.5)
Reaction schemes and rate constants are shown.
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Transient Absorption Studies of the Oxidation of Guanine by
CO
3 Radicals in DNA Duplexes--
Typical transient
absorption spectra of a solution of
d(AACGCGAATTCGCGTT)
duplexes (50 µM) containing 25 mM
S2O
and 300 mM HCO
, 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 µs exhibiting a maximum at 600 nm corresponds to the well known
spectrum of the CO
3 radical (31, 45, 46); at this
HCO
concentration (300 mM), the decay of the SO
4 anion radicals occurs
rapidly (within ~0.7 µs) and is not shown in Fig. 1. The decay of
the CO
3 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
CO
3 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
CO
3 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-R9). 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).

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Fig. 1.
Kinetics of oxidation of guanine in the
duplex d(AACGCGAATTCGCGTT) (50 µM) by CO 3
radicals in air-equilibrated buffer solution containing 25 mM S2O and
300 mM HCO ions (pH
7.5). The CO 3 radicals were indirectly generated (see
text) by SO 4 radicals obtained by the photolysis of
S2O 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 CO 3 radical
anions, and the 315 nm maximum is due to G( H)· radicals. The
lines are shown for visualization only.
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Deprotonation of Guanine Radical Cations--
The
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 s
1). 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 CO
3 Radicals in DNA
Duplexes--
In the absence of DNA, the bimolecular recombination of
two CO
3 radicals occurs, and the rate constant
(k4) of this bimolecular reaction has been
reported (Table I, Reaction 4). Consequently, the decay profile of
CO
3 radical, monitored at 600 nm (Fig. 1), can be described by
mixed first and second order kinetics.
|
(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 CO
3 anion radicals with the DNA duplexes. Defining the initial concentration of carbonate radical anions (at
t = 0) as [CO
3]0, the solution
of Equation 1 yields the following expression for the time dependence
of the CO
3 radical concentration.
|
(Eq. 2)
|
The value of k5' was determined by fitting
Equation 2 to the CO
3 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
M
1 s
1. The rate of oxidation of
dGMP by CO
3 radicals was determined by similar experiments
(data not shown), and the rate constant k6 = 6.6 × 107 M
1
s
1 (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 CO
3 radicals (see
below).

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Fig. 2.
Dependence of the rate constant,
k5', characterizing the decay of
CO 3 radical anions, on the concentration of the
self-complementary d(AACGCGAATTCGCGTT) duplex in air-equilibrated
buffer solutions (pH 7.5) containing 25 mM
S2O and 300 mM
HCO . 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.
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Decay Kinetics of G(
H)· Radicals--
The 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).

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Fig. 3.
Decay kinetics of the 315 nm absorbance of
the guanine radicals, G( H)·, in the self-complementary duplex
d(AAC GCGAATTCGCGTT) (100 µM) in 25 mM
S2O and 300 mM
HCO buffer solution, following excitation with a 308 nm excimer laser pulse (~70 mJ/pulse/cm2).
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Chemical Damage in Double-stranded DNA Induced by CO
3
Radicals--
The rate constant of reaction of SO
4 radicals
with DNA is greater than that of CO
3 radicals (Table I,
Reactions 11 and 5, respectively). The value of
k11 = 5 × 109
M
1 s
1 (Table I) was determined
from the kinetic decay profiles of the SO
4 radical anions
monitored at 445 nm (absorption maximum of SO
4) in solution
containing a 50 µM concentration of the
d(AACGCGAATTCGCGTT) duplex
and 25 mM
S2O
. This constant is
close to the rate constant of the oxidation of dG by SO
4
radicals (k = 2.3 × 109
M
1 s
1) reported by O'Neill and
Davies (37). To minimize the generation of G(
H)· by
SO
4 radicals in the DNA cleavage experiments and to focus on
the reaction of CO
3 radicals with the DNA duplex, a 300 mM concentration of HCO
was selected in these experiments. Under these conditions, the
SO
4 radicals decay predominately via reactions with
HCO
, and their lifetimes are less
than 0.7 µs (data not shown). Thus, reactions of CO
3 with
the DNA duplex are dominant.
DNA Strand Cleavage--
These experiments were performed with the
aim of determining whether all guanines are uniformly damaged after
reaction with CO
3 radical anions. Oxidative guanine base
damage in DNA can be revealed by treatment with hot piperidine
solutions, which gives rise to strand 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 µM
d(AACGCGAATTCGCGTT) duplex,
10 mM S2O
,
and 300 mM HCO
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 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
CO
3 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 one-electron transfer mechanisms more readily with
guanine than with the other three DNA bases.

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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(AACG1CG2AATTCG3C- G4TT)
strands in the duplex form (10 µM) in air-equilibrated
buffer solutions (pH 7.5) containing 10 mM
S2O and 300 mM HCO . 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
G1, G2, G3, and G4 in
the irradiated, self-complementary 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.
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|
 |
DISCUSSION |
Selective Oxidation of Guanines in DNA by CO
3
Radicals--
The selective reactivity of the CO
3 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
CO
3 radicals at pH 7, estimated from the redox potential
E0
(CO
3/CO
) = 1.59 V
versus NHE (51) and the pKa values
of 6.37 (CO2, H2O/HCO
) and 10.25 (HCO
/CO
) (52), and pKa < 0 (HCO
3/CO
3) (46), is ~1.7 V versus NHE. The selective oxidation of guanines by CO
3 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 CO
3 radical anions. The oxidized guanine
derivative 8-oxo-dG, a product of two-electron oxidation of guanine
(50), is further oxidized by CO
3 radicals with a greater rate
constant than in the case of guanine (Table I). This enhanced
reactivity of 8-oxo-dG with CO
3 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 CO
3
Radicals with Guanines--
It is interesting to note that the rate
constant of oxidation of dGMP by CO
3 radical anions
(k6 = 6.6 × 107
M
1 s
1; 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 M
1 s
1 (55), and
1.4 × 109 M
1
s
1 (35), respectively. These results indicate that the
reactivity of CO
3 radicals with guanines is lower than
expected from differences in redox potentials alone. Schindler
et al. (56) have shown that the
CO
3/CO
system is
characterized by a small self-exchange rate constant (k =
0.4 M
1 s
1) and a high internal
reorganization energy that results in a low reactivity of CO
3
in bimolecular outer sphere electron transfer reactions. Furthermore,
in double-stranded DNA the rate constant of oxidation of guanines is
even lower than in the case of free dGMP. This value,
k6 = 1.9 × 107
M
1 s
1 (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 CO
3 radical anion with guanines in double-stranded DNA are
most likely due to site-dependent base pair opening rates (42) and probably to steric hindrance factors as well.
The Long Lifetimes of Guanine Radicals, G(
H)·, in
Double-stranded DNA--
It 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
O
2 radicals (58). Hence, a possible reason for the faster
decay of the G(
H)· radicals in the 2AP-modified duplexes is
the fast reaction of G(
H)· radicals with O
2
radicals. Recently, Candeias and Steenken (59) have reported that the
reaction of G(
H)· with O
2 indeed occurs rapidly with
a rate constant that is nearly diffusion-controlled (~3 × 109 M
1 s
1). In
contrast, the reaction of G(
H)· with O2 (60) is
very slow (k
102
M
1 s
1). A lifetime of
G(
H)· in DNA as long as ~5 s has been reported (48). The
presence or the absence of O
2 could thus be one crucial factor
that determines the lifetime of G(
H)· radicals in
double-stranded DNA.
Multiple Pathways of CO
3 Generation in Biological
Systems--
It is generally accepted that aging, chronic
inflammation, and diverse infectious disorders are associated with an
enhanced production of reactive oxygen species, like superoxide
radicals (O
2), 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
HCO
anions and thus generate the
CO
3 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 HCO
(67)
is associated with the generation of CO
3 radicals by
one-electron oxidation of HCO
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 O
2 radicals in vivo
can result in the formation of the highly reactive and toxic
ONOO
. The combination of ·NO and O
2
occurs with a diffusion-controlled rate constant, k = (6.7-19) × 109 M
1
s
1 (71, 72). That leads to the formation of
ONOO
even at very low concentrations of ·NO and
O
2. Lymar and Hurst (6) have demonstrated that in neutral aqueous solutions, ONOO
rapidly reacts with
CO2 (k = 3 × 104
M
1 s
1) to yield highly unstable
ONOOCO
. Homolytic dissociation of
ONOOCO
occurring on submicrosecond
time scales (73) produces CO
3 and ·NO2
radicals with a cage escape yield of ~0.3 (16, 18).
Hence, because of the relatively high concentrations of
HCO
/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 CO
3 radicals that can
damage not only proteins (1, 12) but also DNA as shown in this report.