INT .
J.
RADIAT . BIOL .,
1992,
VOL .
61,
NO .
1, 15-20
Rapid Communication
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Ionization of polynucleotides and DNA in aqueous solution by 193 nm pulsed laser light : identification of base-derived radicals L. P . CANDEIASt, P. O'NEILL$, G. D. D . JONES$ and S . STEENKENt (Received 1 May 1991 ; revision received 26 June 1991 ; accepted 28 June 1991)
Abstract . Light of 193 nm wavelength ionizes polyA, polyC, polyG, and ss and ds DNA in aqueous solution in a monophotonic process giving hydrated electrons and radicals that result from the radical cations of the bases ; there is spectroscopic evidence for positive charge migration in DNA from adenine to guanine moieties .
It is known that UV radiation damages DNA (Kochevar and Buckley 1990, Cadet and Vigny 1990, Schulte-Frohlinde et al. 1990), of which single- and double-strand breaks are particularly dangerous since they may lead to mutations and cell death . One of the processes leading to light-induced chemical modification of DNA is ionization . The absorption of light of A Z 185 nm occurs almost exclusively (z 99%) at the bases (Kasama et al . 1982, Candeias and Steenken 1991), because they have much higher extinction coefficients (s) than the 2'-deoxyribose-phosphate moiety at these wavelengths (Voet et al. 1963, see also Candeias and Steenken 1991 for the s-values) . In most of the previous studies on the effect of UV radiation on DNA, light of wavelengths : 250 nm has been used (Cadet and Vigny 1990, Schulte-Frohlinde et al . 1990) . The energy provided by one photon of such light (250 nm n 5 eV) is not sufficient to ionize the DNA bases whose (gas phase) ionization potentials are ? 8 eV (Orlov et al. 1976) . Therefore, if ionization is to occur, at least two photons are required Nikogosyan and Letokhov 1983, Schulte-Frohlinde et al . 1990) . In contrast, 193 nm light of energy 6.4 eV per photon is capable of ionizing the DNA bases in a monophotonic process, which is thermodynamically feasible since the solvation of the resulting tMax-Planck-Institut fur Strahlenchemie, D-4330 Mulheim, Germany. $MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD, UK .
ions provides an additional S 3 .5 eV (Patrick and Rahn 1976, Braun et al. 1986) . We have recently shown that with light of 193 nm, ionization is an important photochemical process, and have reported the quantum yields for this reaction for mononucleotides and their components, the bases, (deoxy)ribose, and phosphate (Candeias and Steenken 1990, 1991) . We have now investigated the photoionization of the polynucleotides polyG, polyA, polyC, and polyU, and DNA in its native (double-stranded) and denatured (single-stranded) forms in aqueous solution with the aim of identifying the base transients formed . The relationship between 193 nm ionization and strand break formation is also being investigated at Mulheim (Gorner et al . 1991) . As previously described (Candeias and Steenken 1991), an excimer laser (Lambda Physik EMG 103 MSC) was used which delivered 20 ns pulses of (unfocused) 193 nm light (ArF*) with a power of 3-45 mJ/pulse measured at the position of the cell . The polynucleotides (Pharmacia) and calf thymus DNA (Sigma) were dissolved in water purified with a Millipore Milli-Q system in concentrations such that the optical density (OD) at 193 nm was : 1 .2/ cm, as measured with a Zeiss PMQ spectrophotometer whose A-scale was calibrated to 5 0. 2 nm with the emission lines from a low-pressure Hg lamp. Single-stranded DNA was prepared by heating calf thymus DNA in aqueous solution at 90°C for 10 min followed by rapid chilling in an ice-salt bath . The solutions (pH 7 .0±0.5) were deaerated with argon or saturated with 0 2 and then flowed through the 2 mm (in the direction of the laser light) by 4 mm (in the direction of the analysing light) quartz cells at rates of x 3 ml/min, and photolysed using a pulse repetition rate of 0 .4 Hz . The quantum yields were determined by calibration
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16
L. P. Candeias et al .
with aqueous NaCl solutions of the same OD at 193 nm as that of the solutions of the polynucleotides or DNA ([NaCI] -_ 5 x 10-3 mol dm -3) . The quantum yield for production of electrons from ionization of Cl - was assumed to be the same as that with 185 nm light, reported as 0 . 46 (Dainton and Fowles 1965) . Blank experiments were performed by photolysing water, and no optical signals were observed in the wavelength range studied (200-750 nm), showing that the production of hydrated electrons (e - ) from ionization of water is negligible . On photolysis of deaerated neutral aqueous solutions of polyC, polyA, polyG or DNA (ss and ds) the absorption spectra observed at short times (e.g. 0 . 5µs) after the laser pulse showed a strong and broad band centred at 720 nm, weaker absorptions in the range 300-400 nm, and bleaching at wavelengths around 250 nm . The absorption at 720 nm decayed in deaerated solution in the 100 microsecond time range . The lifetime was drastically reduced by the addition of 0 2 or N 20 . This kinetic behaviour, the quantitative analysis of which gave rate constants for reaction of 0 2 and N 20 in agreement with those reported by Buxton et al. (1988) for eaq, and the shape and 'max of the band observed are clear evidence for the hydrated electron . The transients absorbing at 300-400 nm were not affected by 0 2 and decayed in the millisecond time range . They are assigned (see below) to the (neutral) radicals resulting from the purine and pyrimidine radical cations which deprotonate and/or add water (O'Neill and Davies 1987, Vieira and Steenken 1987a,b, 1990, Candeias and Steenken 1989, 1991, Steenken, 1989, Deeble et al . 1990, Fielden et al. 1991) . The bleaching observed at about 250 nm reflects the depletion of the parent compounds due to destruction of the base chromophores . With polyG, polyA, polyC and DNA (ss and ds) the yield of hydrated electron, as measured by the OD at 650 nm immediately after the laser pulse, was found to vary linearly with the laser intensity, for intensities below 25 mJ per pulse (see Figure 1 and inset of Figure 4) . Above this value the yields levelled off, i .e. saturation effects became apparent . From these results it is evident that the oxidation of polyG, polyA, polyC and DNA (ss and ds) by 193 nm light is a monophotonic process . The quantum yields of photoionization (Op,, Table 1) determined from the slopes of the straight lines compared to that from NaCl ((Dp, = 0 . 46) are ;:z0 . 03-0 . 04, i .e. a few percent efficiency, as previously determined for the mononucleotides (Candeias and Steenken 1991) and polynucleotides (Gorner et al . 1991) . The (Dp, values correlate with the gas phase ionization
potentials JP) of the constituent bases (Orlov et al . 1976), the base with the lowest IP (guanine) giving the highest Ip, (polyG) . Experiments were also performed in which the photoionization of the polynucleotides or of DNA was carried out in oxygen-saturated solution . Under these conditions ([0 2] > [polynucleotide]), the absorption at 720 nm decayed within 0 . 2 its . This is explained by the reaction of eaq with 0 2 (k=1 . 9 x 10 10 dm 3 mol -1 s, Buxton et al . 1988) to give 02'- which does not absorb above 300 nm (Bielski 1978) . The absorption spectra recorded under these conditions, for times > 0 . 2 ps after the pulse and at wavelengths > 300 nm, are similar to those obtained (see Figure 2) upon reaction of the polynucleotides, especially polyA and polyG, with radiolytically produced S0 4 ' -, which is known to react selectively with the bases (O'Neill and Davies 1987) and to produce the corresponding oneelectron-oxidized species (deprotonated radical cations or water adducts, see O'Neill and Davies 1987, Vieira and Steenken 1987a,b, 1990, Candeias
E
C 0 0
0
Figure 1 . Dependence on laser power of the OD at 650 nm (in arbitrary units) measured immediately after the pulse on 193 nm photolysis of deaerated aqueous solutions of polyG (0), polyA (0) and polyC [M] .
Table 1 . Quantum yields of photoionization (cP1) of polynucleotides by 193 nm laser light in neutral aqueous solution at 20±1°C a
OPI
polyG polyA polyC polyU ssDNA dsDNA
0.044 0 .034 0 .029 0 .032b 0.043 0.036
'Estimated error 30% . b Biphotonic ionization ; quantum yield for 22 mJ/pulse .
Photoionization of polynucleotides
0 0
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a
Figure 2 . Absorption spectra recorded on 193 nm laser photolysis of an oxygen-saturated neutral aqueous solution of polyA, 0 . 7µs after the pulse (0) and on reaction of polyA with S04 ' - produced by pulse radiolysis of a deaerated solution at pH 7 . 2 containing 0 . 7 gdm -3 polyA (!` 2 x 10 -3 mol nucleotide/dm -3 ), 2x10 -Z moldm -3 5 2 0 82- , 0 . 4moldm -3 t-butanol, 7 x 10 -3 moldm -3 NaC1O4 and 5 x 10 -3 mol dm -3 phosphate, 25 µs after the pulse (0) . The spectra are normalized to the peak at 330 nm .
and Steenken 1989, Deeble et al . 1990, Hildenbrand 1990) . The similarity of the spectra shows that on photolysis of the polynucleotides in the presence of 0 2 to scavenge eaq , the radicals resulting from the base radical cations are the predominant species
300
400
17
absorbing above 300 nm . However, the spectra are not identical (see Figure 2), from which it is inferred that species other than those resulting from ionization of the bases are also present on this time scale, as observed in the case of 193 nm photolysis of the mononucleotides (Candeias and Steenken 1991) . The results can nevertheless be interpreted in terms of photoionization of the bases of the polynucleotides and DNA (P) being an important reaction induced by 193 nm light . The radical cations produced by the ionization (P' +, equation 1), are suggested to rapidly deprotonate or react with water, based on analogy with the behaviour of the radical cations of mononucleotides (Candeias and Steenken 1990, 1991) . Since the formation of the transients is complete within the 20 ns laser pulse, the lifetime of the excited state leading to ionization (P*) is shorter than 20 ns : Phv1P* s2ons, P .+ +e_ (1) aq The spectrum recorded on photolysis of deaerated aqueous solutions of polyU, shortly after the laser pulse, contained contributions due to ee q and of transients absorbing at > 300 nm, as observed with the other polynucleotides and DNA. However, with polyU an additional transient with 2max = 340 nm was detected (Figure 3) . This transient was found to decay in deaerated solution by first-order kinetics
500
600
700
nm Figure 3 . Absorption spectra recorded on 193 nm laser photolysis of an oxygen-saturated aqueous solution of polyU, 0 . 35 ps (0) and 0-73 ,us ( •) after the pulse . The s-values were obtained by comparison with the OD values measured at 650 nm and taking s(eaq ) at 650 nm to be 15 900dm 3 mol-1 s-1 (from Buxton et al. 1988) and assuming O(e) =I(radical) . Inset (a) Dependence on the square of the laser power of the OD at 650 nm measured immediately after the pulse with a deaerated aqueous solution of polyU . Inset (b) Dependence of the observed rate of decay of the OD at 340 nm, on the oxygen concentration .
L. P. Candeias et al .
18 4000
E C
3000
O O
E
0 0
U
M
E 2000
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0 E
0
5 10 15 20 laser power / mJ/pulse
W
1000
0
ssDNA
I 300
W
400
500
0-
600
•„• 700
X / nm Figure 4. Comparison of the absorption spectrum recorded on 193 nm photolysis of an oxygen saturated solution of ss DNA 5µs after the pulse ( •) with that obtained by addition of the spectra recorded under the same conditions with polyG, polyA and polyC (0) using the following weight factors : polyG 70% ; polyA 5% ; polyC 25% . Inset : dependence on laser power of the OD at 650 nm measured immediately after the pulse with a deaerated solution of ssDNA .
with the rate constant k=2 .3 x 10 5 s -1 . The rate of decay of this species increased linearly with oxygen concentration, giving a rate constant for its reaction with 0 2 of 7 .0 x 108 dm3 mol -1 s -1 (inset b of Figure 3) . From its A max , rate of decay and reactivity with 0 2 this transient is assigned to the triplet state of uracil, which has been observed previously upon excitation with 248 nm light (Gorner 1990) . PolyU differed from the other polynucleotides and DNA also with respect to the dependence of the yield of eay on the laser intensity . The OD (650 nm) measured immediately after the pulse upon photolysis of plyU deaerated solutions increased quadratically with the laser energy, in the energy range below 20 mJ/pulse (inset a of Figure 3) . This shows that the ionization of polyU, even with 193 nm light, is a biphotonic process . The spectrum observed on photolysis of ss DNA in the presence of 0 2 was simulated by weighted averages of the individual spectra recorded on photolysis of polyG, polyA and polyC, also in the presence of 0 2. Two alternatives were considered . In the first, the spectra of polyG, polyA and polyC were weighted according to the products of their Opt values (Table 1) and the c values for the corresponding 2'-deoxymononucleotides (Candeias and Steenken 1991), i .e. 54% G, 28% A, and 18% C .
This resulted in a good fit of the peak at 295 nm, but the agreement between the measured and the computed spectra at A > 320 nm was not satisfactory, particularly at 450-700 nm, where the radical from the adenine moiety absorbs (see Figure 2) . A considerable improvement was achieved by the second method, which consisted in increasing the weight of the oxidized G moiety (which at pH 7 is the deprotonated radical cation of G, see Candeias and Steenken 1989 and references therein) at the expense of the oxidized form of A (the deprotonated radical cation, see O'Neill and Davies 1987 and Steenken 1989), and using a similar contribution of oxidized C as in the first method . The good agreement between the measured and the synthesized spectra (see Figure 4) can be taken as evidence for positive charge transfer from A to G moieties, i .e. electron transfer from G to A . With this interpretation it is assumed that ionization of thymine in DNA does not contribute significantly to the overall spectrum at % > 300 nm . This assumption is not unreasonable for conditions of low laser power if, as found in the case of thymidine (Candeias and Steenken 1991), ionization of this moiety in DNA is biphotonic . In summary, 193 nm light ionizes polyA, polyC and polyG in aqueous solution in a monophotonic process, giving hydrated electrons and radicals that
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Photoionization of polynucleotides result from the radical cations of the bases . The quantum yield of photoionization decreases in the order polyG > polyA > polyC, inversely to that of the gas phase ionization potentials of the corresponding bases. With polyU the ionization is biphotonic . With this polynucleotide, formation of the triplet state and its decay, spontaneous and by reaction with 02, was observed . DNA (ss and ds) is ionized in a monophotonic process and the ionization is tentatively proposed to occur more at the purine, with positive charge migration to guanine, than at the pyrimidine bases . We are presently studying DNA samples of different base-pair composition in order to obtain more quantitative data on preferred sites of ionization and/or charge migration phenomena between bases .
Acknowledgement We thank the British Council for financial support of this work, and Deutscher Akademischer Austauschdienst (DAAD) for a scholarship .
References BIELSKY, B . H . J., 1978, Re-evaluation of the spectral and kinetic properties of HO2 and 02 - free radicals . Photochemistry and Photobiology, 28, 645-649 . BRAUN, M ., FAN, J. Y ., Fuss, W., KOMPA, K . L ., MOLLER, G . and SCHMID, W . E ., 1986, UV Laser ionization spectroscopy and ion photochemistry . Methods of Laser Spectroscopy edited by Y. Prior et al. (Plenum, London), pp . 367-378 . BUXTON, G . V ., GREENSTOCK, C . L ., HELMAN, W . P. and Ross, A. B ., 1988, Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (OH' /0' - ) in aqueous solution . Journal of Physics and Chemistry Reference Data, 17, 513-886. CADET, J. and VIGNY, P ., 1990, The photochemistry of nucleic acids . Bioorganic Photochemistry, edited by H . Morrison (Wiley, New York), vol . 1, pp. 1-272 . CANDEIAS, L . P. and STEENKEN, S ., 1989, Structure and acid-
19
Br - , SO, 2- or OH - ions. Proceedings
of London
of the Royal Society
A, 287, 312-327 .
DEEBLE, D . J ., SCHUCHMANN, M . N ., STEENKEN, S . and VON SONNTAG, C ., 1990, Direct evidence for the formation of thymine radical cations from the reaction of S04 ' - with thymine derivatives : A pulse radiolysis study with optical and conductance detection . Journal of Physical Chemisty, 91, 4138-4144 . FIELDEN, E . M., O'NEILL, P. and STEENKEN, S ., 1991, Chemical reactivity of DNA radicals : a reflection of their redox properties . NATO ARW Early effects of radiation on DNA, edited by E . M . Fielden and P . O'Neill (Springer Verlag, Berlin), vol . 454, pp . 231-247 . GORNER, H., 1990, Transients of uracil and thymine derivatives and the quantum yields of electron ejection and intersystem crossing upon 20 ns photolysis at 248 nm . Photochemisty and Photobiology, 52, 935-948 . GORNER, H ., WALA, M . and SCHULTE-FROHLINDE, D ., 1991, Strand breakage in poly(C), poly(A), single- and double-stranded DNA induced by nanosecond laser excitation at 193 run. Photochemistry and Photobiology (Submitted .) HILDENBRAND, K ., 1990, The SO,* - -induced oxidation of 2'-deoxyuridine-5'-phosphate, uridine-5'-phosphate and thymidine-5'-phosphate . An ESR study in aqueous solution . Z . Naturforsch., 45c, 47-58 . KASAMA, K ., TAKEMATSU, A. and ARAI, S ., 1982, Photochemical reactions of triplet acetone with indole, purine, and pyrimidine derivatives . Journal of Physical Chemistry, 86, 2420-2427 . KOCHEVAR, I . E . and BUCKLEY, L . A ., 1990, Photochemistry of DNA using 193 nm excimer laser radiation . Photochemistry and Photobiology, 51, 527-532 . NIKOGOSYAN, D. N ., 1990, Two quantum UV photochemistry of nucleic acids : Comparison with conventional lowintensity UV photochemistry and radiation chemistry .
International Journal of Radiation Biology, 57, 233-299 . NIKOGOSYAN, D . N . and LETOKHOV, V . S ., 1983, Non-linear laser photophysics, photochemistry and photobiology of nucleic acids . Riv. Nuovo Cimento Soc . Ital. Fis ., Series 3, 6, 1-72 . O'NEILL, P . and DAVIES, S . E ., 1987, Pulse radiolytic study of the interaction S04 ' - with deoxynucleosides . Possible implications for direct energy deposition . International Journal of Radiation Biology, 52, 577-587 . ORLOV, V . M ., SMIRNOV, A . N . and VARSHAVSKY, Y . M., 1976, Ionization potentials and electron-donor ability of nucleic acid bases and their analogues . Tetrahedron
base properties of one-electron-oxidized deoxyguanosine, guanosine, and 1-methylguanosine, Journal of the American Chemical Society, 111, 1094-1099 .
Letters, 4377-4378 . PATRICK, M . H. and RAHN, R . 0 ., 1976, Photochemistry of DNA and polynuceotides . Photo products . In : Wang, S . Y . (Ed .) Photochemisty and Photobiology of Nucleic Acids, vol . II (Academic Press, New York), pp . 35-95 . SCHULTE-FROHLINDE, D ., SIMIC, M . G . and GORNER, H ., 1990,
CANDEIAS, L. P . and STEENKEN, S ., 1990, Ionization of purines and their nucleosides by 193 nm laser photolysis, Abstracts of the Association for Radiation Research Meeting, Oxford, UK, 4-6 April . International Journal of Radiation Biology, 58, 889 . CANDEIAS, L . P . and STEENKEN, S ., 1991, Ionisation of purine nucleosides and nucleotides and their components by 193 nm laser photolysis in aqueous solution . Model studies for oxidative damage of DNA . Journal of the American Chemical Society, in press. DAINTON, F. S . and FowLES, P., 1965, The photolysis of aqueous systems at 1849 A II . Solutions containing C1 - ,
Laser induced strand break formation in DNA and polynucleotides . Photochemisty and Photobiology, 52, 1137-1151 . STEENKEN, S ., 1989, Purine bases, nucleosides and nucleotides : Aqueous solution redox chemistry and transformation reactions of their radical cations and e - and OH adducts . Chemical Reviews, 89, 503-520 . VIEIRA, A. J. S . C . and STEENKEN, S ., 1987a, Pattern of OH radical reaction with N 6, N6 -dimethyladenosine . Production of three isomeric OH adducts and their dehydration and ring-opening reactions . Journal of the American Chemical Society, 109, 7441-7448 .
20
Photoionization of polynucleotides
A. J . S . C . and STEENKEN, S ., 1987b, Pattern of OH radical reaction with 6- and 9-substituted purines . Effect of substituents on the rates and activation parameters of the unimolecular transformation reactions of two isomeric OH adducts . Journal of Physical Chemistry, 91, 4138-4144 . VIEIRA, A . J . S . C. and STEENKEN, S ., 1990, Pattern of OH radical reaction with adenine and its nucleosides and
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VIEIRA,
VOET,
nucleotides . Characterization of two types of isomeric OH adduct and their unimolecular transformation reactions . Journal of the American Chemical Society, 112, 6986-6994 . D ., GRATZER, W . B., Cox, R . A . and DOTY, P., 1963, Absorption spectra of nucleotides, polynucleotides, and nucleic acids in the far ultraviolet . Biopolymers, 1, 193-208 .
J.
RADIAT . BIOL .,
1992,
VOL .
61,
NO .
1, 15-20
Rapid Communication
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Ionization of polynucleotides and DNA in aqueous solution by 193 nm pulsed laser light : identification of base-derived radicals L. P . CANDEIASt, P. O'NEILL$, G. D. D . JONES$ and S . STEENKENt (Received 1 May 1991 ; revision received 26 June 1991 ; accepted 28 June 1991)
Abstract . Light of 193 nm wavelength ionizes polyA, polyC, polyG, and ss and ds DNA in aqueous solution in a monophotonic process giving hydrated electrons and radicals that result from the radical cations of the bases ; there is spectroscopic evidence for positive charge migration in DNA from adenine to guanine moieties .
It is known that UV radiation damages DNA (Kochevar and Buckley 1990, Cadet and Vigny 1990, Schulte-Frohlinde et al. 1990), of which single- and double-strand breaks are particularly dangerous since they may lead to mutations and cell death . One of the processes leading to light-induced chemical modification of DNA is ionization . The absorption of light of A Z 185 nm occurs almost exclusively (z 99%) at the bases (Kasama et al . 1982, Candeias and Steenken 1991), because they have much higher extinction coefficients (s) than the 2'-deoxyribose-phosphate moiety at these wavelengths (Voet et al. 1963, see also Candeias and Steenken 1991 for the s-values) . In most of the previous studies on the effect of UV radiation on DNA, light of wavelengths : 250 nm has been used (Cadet and Vigny 1990, Schulte-Frohlinde et al . 1990) . The energy provided by one photon of such light (250 nm n 5 eV) is not sufficient to ionize the DNA bases whose (gas phase) ionization potentials are ? 8 eV (Orlov et al. 1976) . Therefore, if ionization is to occur, at least two photons are required Nikogosyan and Letokhov 1983, Schulte-Frohlinde et al . 1990) . In contrast, 193 nm light of energy 6.4 eV per photon is capable of ionizing the DNA bases in a monophotonic process, which is thermodynamically feasible since the solvation of the resulting tMax-Planck-Institut fur Strahlenchemie, D-4330 Mulheim, Germany. $MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD, UK .
ions provides an additional S 3 .5 eV (Patrick and Rahn 1976, Braun et al. 1986) . We have recently shown that with light of 193 nm, ionization is an important photochemical process, and have reported the quantum yields for this reaction for mononucleotides and their components, the bases, (deoxy)ribose, and phosphate (Candeias and Steenken 1990, 1991) . We have now investigated the photoionization of the polynucleotides polyG, polyA, polyC, and polyU, and DNA in its native (double-stranded) and denatured (single-stranded) forms in aqueous solution with the aim of identifying the base transients formed . The relationship between 193 nm ionization and strand break formation is also being investigated at Mulheim (Gorner et al . 1991) . As previously described (Candeias and Steenken 1991), an excimer laser (Lambda Physik EMG 103 MSC) was used which delivered 20 ns pulses of (unfocused) 193 nm light (ArF*) with a power of 3-45 mJ/pulse measured at the position of the cell . The polynucleotides (Pharmacia) and calf thymus DNA (Sigma) were dissolved in water purified with a Millipore Milli-Q system in concentrations such that the optical density (OD) at 193 nm was : 1 .2/ cm, as measured with a Zeiss PMQ spectrophotometer whose A-scale was calibrated to 5 0. 2 nm with the emission lines from a low-pressure Hg lamp. Single-stranded DNA was prepared by heating calf thymus DNA in aqueous solution at 90°C for 10 min followed by rapid chilling in an ice-salt bath . The solutions (pH 7 .0±0.5) were deaerated with argon or saturated with 0 2 and then flowed through the 2 mm (in the direction of the laser light) by 4 mm (in the direction of the analysing light) quartz cells at rates of x 3 ml/min, and photolysed using a pulse repetition rate of 0 .4 Hz . The quantum yields were determined by calibration
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16
L. P. Candeias et al .
with aqueous NaCl solutions of the same OD at 193 nm as that of the solutions of the polynucleotides or DNA ([NaCI] -_ 5 x 10-3 mol dm -3) . The quantum yield for production of electrons from ionization of Cl - was assumed to be the same as that with 185 nm light, reported as 0 . 46 (Dainton and Fowles 1965) . Blank experiments were performed by photolysing water, and no optical signals were observed in the wavelength range studied (200-750 nm), showing that the production of hydrated electrons (e - ) from ionization of water is negligible . On photolysis of deaerated neutral aqueous solutions of polyC, polyA, polyG or DNA (ss and ds) the absorption spectra observed at short times (e.g. 0 . 5µs) after the laser pulse showed a strong and broad band centred at 720 nm, weaker absorptions in the range 300-400 nm, and bleaching at wavelengths around 250 nm . The absorption at 720 nm decayed in deaerated solution in the 100 microsecond time range . The lifetime was drastically reduced by the addition of 0 2 or N 20 . This kinetic behaviour, the quantitative analysis of which gave rate constants for reaction of 0 2 and N 20 in agreement with those reported by Buxton et al. (1988) for eaq, and the shape and 'max of the band observed are clear evidence for the hydrated electron . The transients absorbing at 300-400 nm were not affected by 0 2 and decayed in the millisecond time range . They are assigned (see below) to the (neutral) radicals resulting from the purine and pyrimidine radical cations which deprotonate and/or add water (O'Neill and Davies 1987, Vieira and Steenken 1987a,b, 1990, Candeias and Steenken 1989, 1991, Steenken, 1989, Deeble et al . 1990, Fielden et al. 1991) . The bleaching observed at about 250 nm reflects the depletion of the parent compounds due to destruction of the base chromophores . With polyG, polyA, polyC and DNA (ss and ds) the yield of hydrated electron, as measured by the OD at 650 nm immediately after the laser pulse, was found to vary linearly with the laser intensity, for intensities below 25 mJ per pulse (see Figure 1 and inset of Figure 4) . Above this value the yields levelled off, i .e. saturation effects became apparent . From these results it is evident that the oxidation of polyG, polyA, polyC and DNA (ss and ds) by 193 nm light is a monophotonic process . The quantum yields of photoionization (Op,, Table 1) determined from the slopes of the straight lines compared to that from NaCl ((Dp, = 0 . 46) are ;:z0 . 03-0 . 04, i .e. a few percent efficiency, as previously determined for the mononucleotides (Candeias and Steenken 1991) and polynucleotides (Gorner et al . 1991) . The (Dp, values correlate with the gas phase ionization
potentials JP) of the constituent bases (Orlov et al . 1976), the base with the lowest IP (guanine) giving the highest Ip, (polyG) . Experiments were also performed in which the photoionization of the polynucleotides or of DNA was carried out in oxygen-saturated solution . Under these conditions ([0 2] > [polynucleotide]), the absorption at 720 nm decayed within 0 . 2 its . This is explained by the reaction of eaq with 0 2 (k=1 . 9 x 10 10 dm 3 mol -1 s, Buxton et al . 1988) to give 02'- which does not absorb above 300 nm (Bielski 1978) . The absorption spectra recorded under these conditions, for times > 0 . 2 ps after the pulse and at wavelengths > 300 nm, are similar to those obtained (see Figure 2) upon reaction of the polynucleotides, especially polyA and polyG, with radiolytically produced S0 4 ' -, which is known to react selectively with the bases (O'Neill and Davies 1987) and to produce the corresponding oneelectron-oxidized species (deprotonated radical cations or water adducts, see O'Neill and Davies 1987, Vieira and Steenken 1987a,b, 1990, Candeias
E
C 0 0
0
Figure 1 . Dependence on laser power of the OD at 650 nm (in arbitrary units) measured immediately after the pulse on 193 nm photolysis of deaerated aqueous solutions of polyG (0), polyA (0) and polyC [M] .
Table 1 . Quantum yields of photoionization (cP1) of polynucleotides by 193 nm laser light in neutral aqueous solution at 20±1°C a
OPI
polyG polyA polyC polyU ssDNA dsDNA
0.044 0 .034 0 .029 0 .032b 0.043 0.036
'Estimated error 30% . b Biphotonic ionization ; quantum yield for 22 mJ/pulse .
Photoionization of polynucleotides
0 0
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a
Figure 2 . Absorption spectra recorded on 193 nm laser photolysis of an oxygen-saturated neutral aqueous solution of polyA, 0 . 7µs after the pulse (0) and on reaction of polyA with S04 ' - produced by pulse radiolysis of a deaerated solution at pH 7 . 2 containing 0 . 7 gdm -3 polyA (!` 2 x 10 -3 mol nucleotide/dm -3 ), 2x10 -Z moldm -3 5 2 0 82- , 0 . 4moldm -3 t-butanol, 7 x 10 -3 moldm -3 NaC1O4 and 5 x 10 -3 mol dm -3 phosphate, 25 µs after the pulse (0) . The spectra are normalized to the peak at 330 nm .
and Steenken 1989, Deeble et al . 1990, Hildenbrand 1990) . The similarity of the spectra shows that on photolysis of the polynucleotides in the presence of 0 2 to scavenge eaq , the radicals resulting from the base radical cations are the predominant species
300
400
17
absorbing above 300 nm . However, the spectra are not identical (see Figure 2), from which it is inferred that species other than those resulting from ionization of the bases are also present on this time scale, as observed in the case of 193 nm photolysis of the mononucleotides (Candeias and Steenken 1991) . The results can nevertheless be interpreted in terms of photoionization of the bases of the polynucleotides and DNA (P) being an important reaction induced by 193 nm light . The radical cations produced by the ionization (P' +, equation 1), are suggested to rapidly deprotonate or react with water, based on analogy with the behaviour of the radical cations of mononucleotides (Candeias and Steenken 1990, 1991) . Since the formation of the transients is complete within the 20 ns laser pulse, the lifetime of the excited state leading to ionization (P*) is shorter than 20 ns : Phv1P* s2ons, P .+ +e_ (1) aq The spectrum recorded on photolysis of deaerated aqueous solutions of polyU, shortly after the laser pulse, contained contributions due to ee q and of transients absorbing at > 300 nm, as observed with the other polynucleotides and DNA. However, with polyU an additional transient with 2max = 340 nm was detected (Figure 3) . This transient was found to decay in deaerated solution by first-order kinetics
500
600
700
nm Figure 3 . Absorption spectra recorded on 193 nm laser photolysis of an oxygen-saturated aqueous solution of polyU, 0 . 35 ps (0) and 0-73 ,us ( •) after the pulse . The s-values were obtained by comparison with the OD values measured at 650 nm and taking s(eaq ) at 650 nm to be 15 900dm 3 mol-1 s-1 (from Buxton et al. 1988) and assuming O(e) =I(radical) . Inset (a) Dependence on the square of the laser power of the OD at 650 nm measured immediately after the pulse with a deaerated aqueous solution of polyU . Inset (b) Dependence of the observed rate of decay of the OD at 340 nm, on the oxygen concentration .
L. P. Candeias et al .
18 4000
E C
3000
O O
E
0 0
U
M
E 2000
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0 E
0
5 10 15 20 laser power / mJ/pulse
W
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ssDNA
I 300
W
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500
0-
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X / nm Figure 4. Comparison of the absorption spectrum recorded on 193 nm photolysis of an oxygen saturated solution of ss DNA 5µs after the pulse ( •) with that obtained by addition of the spectra recorded under the same conditions with polyG, polyA and polyC (0) using the following weight factors : polyG 70% ; polyA 5% ; polyC 25% . Inset : dependence on laser power of the OD at 650 nm measured immediately after the pulse with a deaerated solution of ssDNA .
with the rate constant k=2 .3 x 10 5 s -1 . The rate of decay of this species increased linearly with oxygen concentration, giving a rate constant for its reaction with 0 2 of 7 .0 x 108 dm3 mol -1 s -1 (inset b of Figure 3) . From its A max , rate of decay and reactivity with 0 2 this transient is assigned to the triplet state of uracil, which has been observed previously upon excitation with 248 nm light (Gorner 1990) . PolyU differed from the other polynucleotides and DNA also with respect to the dependence of the yield of eay on the laser intensity . The OD (650 nm) measured immediately after the pulse upon photolysis of plyU deaerated solutions increased quadratically with the laser energy, in the energy range below 20 mJ/pulse (inset a of Figure 3) . This shows that the ionization of polyU, even with 193 nm light, is a biphotonic process . The spectrum observed on photolysis of ss DNA in the presence of 0 2 was simulated by weighted averages of the individual spectra recorded on photolysis of polyG, polyA and polyC, also in the presence of 0 2. Two alternatives were considered . In the first, the spectra of polyG, polyA and polyC were weighted according to the products of their Opt values (Table 1) and the c values for the corresponding 2'-deoxymononucleotides (Candeias and Steenken 1991), i .e. 54% G, 28% A, and 18% C .
This resulted in a good fit of the peak at 295 nm, but the agreement between the measured and the computed spectra at A > 320 nm was not satisfactory, particularly at 450-700 nm, where the radical from the adenine moiety absorbs (see Figure 2) . A considerable improvement was achieved by the second method, which consisted in increasing the weight of the oxidized G moiety (which at pH 7 is the deprotonated radical cation of G, see Candeias and Steenken 1989 and references therein) at the expense of the oxidized form of A (the deprotonated radical cation, see O'Neill and Davies 1987 and Steenken 1989), and using a similar contribution of oxidized C as in the first method . The good agreement between the measured and the synthesized spectra (see Figure 4) can be taken as evidence for positive charge transfer from A to G moieties, i .e. electron transfer from G to A . With this interpretation it is assumed that ionization of thymine in DNA does not contribute significantly to the overall spectrum at % > 300 nm . This assumption is not unreasonable for conditions of low laser power if, as found in the case of thymidine (Candeias and Steenken 1991), ionization of this moiety in DNA is biphotonic . In summary, 193 nm light ionizes polyA, polyC and polyG in aqueous solution in a monophotonic process, giving hydrated electrons and radicals that
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Photoionization of polynucleotides result from the radical cations of the bases . The quantum yield of photoionization decreases in the order polyG > polyA > polyC, inversely to that of the gas phase ionization potentials of the corresponding bases. With polyU the ionization is biphotonic . With this polynucleotide, formation of the triplet state and its decay, spontaneous and by reaction with 02, was observed . DNA (ss and ds) is ionized in a monophotonic process and the ionization is tentatively proposed to occur more at the purine, with positive charge migration to guanine, than at the pyrimidine bases . We are presently studying DNA samples of different base-pair composition in order to obtain more quantitative data on preferred sites of ionization and/or charge migration phenomena between bases .
Acknowledgement We thank the British Council for financial support of this work, and Deutscher Akademischer Austauschdienst (DAAD) for a scholarship .
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DEEBLE, D . J ., SCHUCHMANN, M . N ., STEENKEN, S . and VON SONNTAG, C ., 1990, Direct evidence for the formation of thymine radical cations from the reaction of S04 ' - with thymine derivatives : A pulse radiolysis study with optical and conductance detection . Journal of Physical Chemisty, 91, 4138-4144 . FIELDEN, E . M., O'NEILL, P. and STEENKEN, S ., 1991, Chemical reactivity of DNA radicals : a reflection of their redox properties . NATO ARW Early effects of radiation on DNA, edited by E . M . Fielden and P . O'Neill (Springer Verlag, Berlin), vol . 454, pp . 231-247 . GORNER, H., 1990, Transients of uracil and thymine derivatives and the quantum yields of electron ejection and intersystem crossing upon 20 ns photolysis at 248 nm . Photochemisty and Photobiology, 52, 935-948 . GORNER, H ., WALA, M . and SCHULTE-FROHLINDE, D ., 1991, Strand breakage in poly(C), poly(A), single- and double-stranded DNA induced by nanosecond laser excitation at 193 run. Photochemistry and Photobiology (Submitted .) HILDENBRAND, K ., 1990, The SO,* - -induced oxidation of 2'-deoxyuridine-5'-phosphate, uridine-5'-phosphate and thymidine-5'-phosphate . An ESR study in aqueous solution . Z . Naturforsch., 45c, 47-58 . KASAMA, K ., TAKEMATSU, A. and ARAI, S ., 1982, Photochemical reactions of triplet acetone with indole, purine, and pyrimidine derivatives . Journal of Physical Chemistry, 86, 2420-2427 . KOCHEVAR, I . E . and BUCKLEY, L . A ., 1990, Photochemistry of DNA using 193 nm excimer laser radiation . Photochemistry and Photobiology, 51, 527-532 . NIKOGOSYAN, D. N ., 1990, Two quantum UV photochemistry of nucleic acids : Comparison with conventional lowintensity UV photochemistry and radiation chemistry .
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Letters, 4377-4378 . PATRICK, M . H. and RAHN, R . 0 ., 1976, Photochemistry of DNA and polynuceotides . Photo products . In : Wang, S . Y . (Ed .) Photochemisty and Photobiology of Nucleic Acids, vol . II (Academic Press, New York), pp . 35-95 . SCHULTE-FROHLINDE, D ., SIMIC, M . G . and GORNER, H ., 1990,
CANDEIAS, L. P . and STEENKEN, S ., 1990, Ionization of purines and their nucleosides by 193 nm laser photolysis, Abstracts of the Association for Radiation Research Meeting, Oxford, UK, 4-6 April . International Journal of Radiation Biology, 58, 889 . CANDEIAS, L . P . and STEENKEN, S ., 1991, Ionisation of purine nucleosides and nucleotides and their components by 193 nm laser photolysis in aqueous solution . Model studies for oxidative damage of DNA . Journal of the American Chemical Society, in press. DAINTON, F. S . and FowLES, P., 1965, The photolysis of aqueous systems at 1849 A II . Solutions containing C1 - ,
Laser induced strand break formation in DNA and polynucleotides . Photochemisty and Photobiology, 52, 1137-1151 . STEENKEN, S ., 1989, Purine bases, nucleosides and nucleotides : Aqueous solution redox chemistry and transformation reactions of their radical cations and e - and OH adducts . Chemical Reviews, 89, 503-520 . VIEIRA, A. J. S . C . and STEENKEN, S ., 1987a, Pattern of OH radical reaction with N 6, N6 -dimethyladenosine . Production of three isomeric OH adducts and their dehydration and ring-opening reactions . Journal of the American Chemical Society, 109, 7441-7448 .
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A. J . S . C . and STEENKEN, S ., 1987b, Pattern of OH radical reaction with 6- and 9-substituted purines . Effect of substituents on the rates and activation parameters of the unimolecular transformation reactions of two isomeric OH adducts . Journal of Physical Chemistry, 91, 4138-4144 . VIEIRA, A . J . S . C. and STEENKEN, S ., 1990, Pattern of OH radical reaction with adenine and its nucleosides and
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