the carbonate radical is a site-selective oxidizing agent of

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

From the Chemistry Department and Radiation and Solid State Laboratory,

31 Washington Place, New York University, New York, New York 10003-5180

Running title: Carbonate Radical Is a Site-Selective Oxidizing Agent

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

JBC Papers in Press. Published on April 24, 2001 as Manuscript M101131200 by guest on February 12, 2018

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SUMMARY

The carbonate anion radical (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 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 one-electron oxidation of the bicarbonate

anions (HCO3-) with sulfate radical anions (SO4.-) which, 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 312 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 M-1s-1. The decay of the CO3.- anions and the formation of G(-

H). radicals are correlated with one another on the millisecond time scale, while 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,

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showing that strand cleavage occurs at the guanine sites only. The biological implications of

these oxidative processes are discussed.

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 (CO3.-)1 from bicarbonate (HCO3-) or CO2 can 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 S-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


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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 peroxynitrite (ONOO-), but enhances the formation of 8-nitroguanine (8-

nitro-G), alkali- and Fpg-labile DNA lesions (23-25). 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). While 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 CO3.- 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 (λmax ~ 600 nm), while the

concomitant formation of guanine neutral radicals, G(-H)., was identified by their characteristic


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narrow absorption band at 312 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.

EXPERIMENTAL PROCEDURES

Materials — 2’-deoxyguanosine 5’-monophospate (dGMP), 8-oxo-7,8-dihydro-2’-

deoxyguanosine (8-oxo-dG), and all inorganic salts (>99% purity) were obtained from Sigma-

Aldrich Fine Chemicals, 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 oligonuclotide was purified by reversed phase

HPLC, 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 oC 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 (~7 ns response time) described earlier (27-

30). Briefly, this system consists of a pulsed Lambda Physik EMG 160 MSC XeCl excimer laser


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(308 nm, full width at half maximum pulse width ~ 12 ns, energy ~ 70 mJ pulse-1 cm-2, 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 photodisscociation of persulfate anions (31,33).

While persulfate ions exhibit neglible 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 time-resolved pulse radiolysis or flash photolysis experiments

has been previously employed by Steenken et al.(34-36), O’Neill et al.(37), and Hildebrandt et

al.(38), to study the kinetics of reactions of the SO4.- 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 1 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 oC.

Pseudo-first order rate constants (kn’) of the radical (R.) + substrate (S) reactions, and the


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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’ vs [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 (Pharmacia, Piscataway, NJ) and [γ-32P]ATP (New

England Nuclear, Boston, MA) 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 β-mercaptoethanol and was heated for 30 min at 90 oC 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 (Bio-Rad,

Hercules, CA).

RESULTS

Duplex design — The duplex formed by the self-complementary 16-mer

d(AACG1CG2AATTCG3CG4TT) sequence contains the 12-mer d(CG1CG2AATTCG3CG4) core. The latter


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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 oC 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.- radicals. The 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 (reaction 1,

Table 1) with a quantum yield of 0.55 (33). The SO4.- radicals exhibit transient absorption

bands at 445 nm with an exctinction coefficients of 1600 M-1cm-1 (44). In pure aqueous buffer

solutions, the SO4.- radical anions decay mostly via bimolecular recombination processes

(reaction 2, Table 1). 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 M-1cm-1 (45). This effect


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is attributed to the reduction of SO4.- by HCO3- with the formation of CO3.- (reaction 3,

Table 1); 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 (reaction 4, Table 1) 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 1; 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

Duplexes — Typical transient absorption spectra of a solution of d(AACGCGAATTCGCGTT)

duplexes (50 µM) containing 25 mM S2O82- and 300 mM HCO3-, recorded at various delay

times (∆t) after 308 nm laser pulse excitation, are shown in Figure 1. The transient absorption

spectrum recorded at ∆t = 60 µs 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 µs), and is not shown in Figure

1. The decay of the CO3.- absorption band is accompanied by the rise of another, narrow

transient absorption band centered at 310 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).


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(34). Thus, the carbonate radical is a site-selective one-electron oxidant of guanine residues in

double-stranded DNA (reaction 5, Table 1). 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 1, reactions 6 - 9). However, the rate constant of oxidation of 8-oxo-dG is

greater by a factor of ~ 10 (Table 1, reaction 10) than that of dGMP (36).

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 ESR techniques upon oxidation of double-stranded DNA in

aqueous solutions at room temperature (48,49). Hence, the guanine radicals observed on

millisecond time scales (Figure 1) are neutral radicals, G(-H)., rather than G.+ radical cations.

Kinetics of guanine oxidation by CO3.- radicals in DNA duplexes. In the absence of DNA,

the bimolecular recombination of two CO3.- radicals occurs and the rate constant, k4, of this


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bimolecular reaction has been reported (see, Table 1, reaction 4). Consequently, the decay profile

of CO3.- radical, monitored at 600 nm (Figure 1), can be described by mixed first- and second-

order kinetics:

-d[CO3.-]/dt = k5’ [CO3.-] + 2k4 [CO3.-]2

Equation 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 Eq. 1 yields the following expression for the time dependence of the CO3.-

radical concentration:

[CO3.-]t = k5’ [CO3.-]0 /{(k5’ + 2k4 [CO3.-]0) exp (k5’ t) - 2k4 [CO3.-]0}

Equation 2

The value of k5’ was determined by fitting Eq. 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 1). As expected, k5’ increases linearly with the concentration of the DNA

duplex (Figure 2), yielding a value of k5 = 1.9×107 M-1 s-1. The rate of oxidation of dGMP


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by CO3.- radicals was determined by similar experiments (data not shown), and the rate constant

k6 = 6.6×107 M-1 s-1 (Table 1). 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). radicals. The decay of the G(-H). radicals in the double stranded

oligonucleotide is shown on two different time scales in Figure 3. On the < 100 ms time scale

(insert, Figure 3), 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 (Figure 3).

Chemical damage in double-stranded DNA induced by CO3.- radicals. The rate constant of

reaction of SO4.- radicals with DNA is greater than that of CO3.- radicals (reactions 11 and 5,

respectively, Table 1). The value of k11 = 5×109 M-1 s-1 (Table 1) was determined from the

kinetic decay profiles of the SO4.- radical anions monitored at 445 nm (absorption maximum of

SO4.- ) in solution containing a 50 µM concentration of the d(AACGCGAATTCGCGTT)

duplex and 25 mM S2O82-. This constant is close to the rate constant of the oxidation of dG by

SO4.- radicals (k = 2.3×109 M-1 s-1) reported by O’Neill and Davies (37). In order to

minimize the generation of G(-H). by SO4.- radicals in the DNA cleavage experiments and to


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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 µs (data not

shown). Thus, reactions of CO3.- 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 CO3.- 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

S2O82- and 300 mM HCO3- with 308 nm excimer laser pulses, are depicted in the gel autoraodigraph

in Figure 4 (Panel A). 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 while in lanes 4 and

6 the dosage was ~ 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


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predominantly at the four different guanines in the self-complementary oligonucleotide duplex.

The relative cleavage efficiencies at the different guanines are shown in panel B, Figure 4. The

probability of strand cleavage at these sites differs, being higher at the guanines G1 and G2

closer to the ends of the oligonucleotide than at sites G2 and G3 that 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 per molecule (0.4 mJ/cm2 dosage). A the higher dosage, the fraction of cleaved

DNA strands increased to 40-45%. Nevertheless, strand cleavage at sites other than guanine

remains small (Figure 4, Panel C). 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 one-electron transfer

mechanisms more readily with guanine than with the other three DNA bases.

DISCUSSION

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.-


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/CO32-) = 1.59 V vs 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 vs NHE. The selective oxidation of

guanines by CO3.- radicals is thus thermodynamically feasible. Since the redox potential of free

dG, E7[dG(-H)./dG] = 1.29 V vs NHE, is smaller than the redox potential of dA, E7[dA(-H).

/dA] = 1.42 V vs NHE, and that of the pyrimidine bases, E7 = 1.7 V (dT) and 1.6 V (dC) vs 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 1). This enhanced reactivity of 8-oxo-dG with CO3.- radicals is correlated with

its lower redox potential, E7 = 0.74 V vs NHE (36), which is smaller than the redox potentials of

the other nucleosides discussed.

Rate constants of interaction of the CO3.- radicals with guanines. It is interesting to note

that the rate constant of oxidation of dGMP by CO3.- radical anions (k6 = 6.6×107 M-1 s-1,

Table 1) 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 vs NHE, ref. 53), and thioanisole (E0 = 1.45

V vs NHE, ref. 54), oxidize dGMP or dG with rate constants of 1.7×109 M-1s-1 (55), and


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1.4×109 M-1s-1 (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 M-1s-1), and a high internal reorganization energy that results in a low

reactivity of CO3.- 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 1), is averaged over the four different

guanine sites G1, G2, G3, and G4. The cleavage efficiencies (Figure 4, Panel B) 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-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 (Figure 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


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d(AACGCGAATTCGCGTT) duplex (Figure 3) is orders of magnitudes longer than in

oligonucleotide duplexes in which these radicals are generated by one-electron transfer reactions

from G 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 due to 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 2AP

modified 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 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 absence of O2.- could thus be one

crucial factor which determines the lifetime of G(-H). radicals in double-stranded DNA.

Multiple pathways of CO3.- 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 (O2.-), hydrogen peroxide


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(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 (SOD1)

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 results

in the formation of the highly reactive and toxic peroxynitrite (ONOO-). The combination of

.NO and O2.- 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 O2.-

. Lymar and Hurst (6) have demonstrated that in neutral aqueous solutions, ONOO- rapidly


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reacts with CO2 (k = 3×104 M-1 s-1) to yield a highly unstable nitrosoperoxycarbonate anion

(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, due to the relatively high concentrations of 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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Footnotes

§This work has been supported by the National Science Foundation, Grant CHE-9700429

and by a grant from the Kresge Foundation.

*To whom correspondence should be addressed at: Chemistry Department and Radiation and

Solid State Laboratory, 31 Washington Place, New York University, New York, New York

10003-5180. Tel.: (212) 998 8456; Fax: (212) 998 8421; e-mail: [email protected]

1 The abbreviations used are: CO3. ,̄ carbonate radical anion; HCO3-, bicarbonate anion; 8-

oxo-dG, 8-oxo-7,8-dihydro-2’-deoxyguanosine; ONOOCO2-, nitrosoperoxycarbonate anion;

ONOO-, peroxynitrite; SO4.- sulfate radical; S2O82-, persulfate; 8-nitro-G, 8-nitroguanine;

8-nitro-dG, 8-nitro-2’-deoxyguanosine; G, guanine; dG, 2’-deoxyguanosine; dGMP, 2’-

deoxyguanosine 5’-monophospate; Fpg, formamidopyrimidine glycosylase; .NO, nitric oxide.


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Figure Legends

Figure 1. Kinetics of oxidation of guanine in the duplex d(AACGCGAATTCGCGTT) (50

µM) 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, full width at half maximum = 12 ns). 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 to G(-H). radicals. The lines are shown for

visualization only.

Figure 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


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air-equilibrated 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 5 individual laser flash photolysis experiments.

Figure 3. Decay kinetics of the 315 nm absorbance of the guanine radicals, G(-H)., in the

self-complementary duplex d(AACGCGAATTCGCGTT) (100 µM) in 25 mM S2O82- and

300 mM HCO3- buffer solution, following excitation with a 308 nm excimer laser pulse (~ 70

mJ/pulse/cm2, fwhm ~ 12 ns).

Figure 4. Gel electrophoresis patterns of irradiated, hot-piperidine oligonucleotide strands.

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 5’-

32P-end-labeled d(AACG1CG2AATTCG3CG4TT) strands in the duplex form (10 µM) 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 (1M, 90 oC) for 30

min and loaded onto a polyacrylamide gel. Panel 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 (integrated dosage received by the sample,

0.5 and 4 J/cm2, respectively): Irradiated duplex (without piperidine treatment); Lanes 4 and 6


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(integrated dosage received by the sample, 0.5 and 4 J/cm2, respectively): Irradiated duplex

(after hot piperidine treatment). Panel 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 5’- 32P end-labeled

d(AACG1CG2AATTCG3CG4TT) duplex. Panel C: Total fraction of strands cleaved at the dosage of 4 J/cm2,

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 (Panels B and C)

obtained in 3 different photocleavage experiments.

Table. 1. Oxidation of the d(AACG1CG2AATTCG3CG4TT) duplex by CO3.- radicals in

aqueous buffer solutions (pH 7.5). Reaction schemes and rate constants.

No Reaction k (M-1s-1) Ref

1S2O82- + hν → 2 SO4.- λ308 = 0.55 (33)


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2SO4.- + SO4.- → S2O82- k2 = (1.1±0.1)×109

k2 = 1.35×109

this work

(33)

3SO4.- + HCO3- → SO42- + CO3.-

k3 = (4.6±0.5)×106

k3 = 9.1×106

k3 = 2.8×106

this work

(31)

(32)

4CO3.- + CO3.- → CO42- + CO2 k4 = (1.3±0.1)×107

k4 = 2×107

this work

(45)

5a CO3.- + d[G-oligo] → CO32- + d[G(-H).-

oligo]

k5 = (1.9±0.2)×107this work

6a CO3.- + dGMP → CO32- + dGMP(-H). k6 = (6.6±0.7)×107this work

7b CO3.- + dAMP → k7 ≤ 2.8×106this work

8b CO3.- + dCMP → k8 ≤ 1.9×106this work

9a CO3.- + dTMP → k9 ≤ 1.7×106this work

10a CO3.- + 8-oxo-dG → CO32- + 8-oxo-G(-H). k10 = (7.9±0.8)×108this work


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11b SO4.- + d[G-oligo] → SO42- + d[G(-H).-

oligo]

k11 = (9.5±0.9)×109this 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.


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Vladimir Shafirovich, Alexander Dourandin, Weidong Huang and Nicholas E Geacintovoligonucleotides

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded

published online April 24, 2001J. Biol. Chem.

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