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Luminescence of DNA excited in the vacuum ultraviolet
28 September1990
Volume173,number1
Luminescence of DNA excited in the vacuum ultraviolet B. Brocklehurst a, A. Hopkirkb and I.H. Munro b ’ Chemistry Department, Universityof Smeld, Sh@eld S3 7Hl? UK b SERC, Daresbury L&oratory,
Warrington WA44AD, UK
Received 1 June 1990
Pulsed vacuum ultravioletradiation has been used to exciteDNAin the energyrange12to 30eV.Time-resolved single-photon countingshowsnon-exponential luminescence decays over some hundreds of nanoseconds: the application of a magnetic field demonstrates the roleof radical-ionrecombinationand,at higherenergies,of fissionintotwotripletspecies.
1. Introduction The effects of ionising radiation are complicated by the size distribution of the spurs - groups of radicals, ion pairs, excited states, etc. The use of the vae uum ultraviolet (WV) simplifies elucidation of the primary processes: e.g., a 10-I 5 eV photon produces a single ion pair, 20-40 eV two ion pairs or an ion pair and an excited molecule. Time-resolved luminescence and magnetic field effects are sensitive probes of subsequent processes [ 11. Most previous work of this type [l-3] has concentrated on hydrocarbons because the radical ions produced initially do not undergo further reactions before recombination: in compounds of oxygen or nitrogen, ion-molecule reactions and dissociative at&hment usually produce neutral radicals and recombination does not lead to excited states. However, we now report that dry DNA irradiated in the WV Iuminesces on a time scale much longer than that of near UV excitation and that field effects can be observed.
2. Experimental The apparatus has been described previously [ 3,4]. When operated in single-buch mode, the Synchrotron Radiation Source at Daresbury provides 200 ps pulses every 320 ns with a continuous spectrum [ 5 1. A Seya monochromator (station 3.1) with two in0009-2614/90/$03.50
terchangeable gratings was used, giving intensities of the order of 10s-log photons per second at the sample. Time-resolved single-photon counting was used to measure the luminescence decays. Data were collected in separate sections of an MCA over several cycles in which an applied field (0.135 T) alternated with zero field for periods of 100 s. Salmon testes DNA (Sigma) was used as received. The results shown are from a single run with the dry material; similar results have been obtained with DNA plated from aqueous solution. Since no windows could be used, the samples were pumped down to c 100~ Torr over a period of at least an hour, and kept in vacua over,several hours during the experiment. Significant water loss from the surface layers probably occurred during this period, leading to a contraction of the helix. During the course of the experiment the pulse-shape changed (fig. 1): the field effect showed smaller changes but the qualitative results were not affected, the results in fig. 3 were obtained by alternating between higher and lower energies. Drying or radiation damage may be responsible for the changes. However, the same striking change in the field effect with energy was observed both early and late in the run.
3. Results and discussion Comparison with crystalline sodium salicylate gave a very rough estimate of 0.003 for the luminescence
0 1990 - Elsevier Science Publishers B.V. (North-Holland)
129
Volume 173, number 1
CHEMICAL PHYSICS LETTERS
l-00
O*lO
0.01
0
50
100
150 t/ns
Fig. 1.Norma&xl luminescencedecay curves for dry DNA measuredduring a single run; (a) excited by 15 eV photons (first
28 September 1990
yield in the VUV; since yields in the near UV are also low [ 61, the inefficiency is in the final luminescence step rather than in the processesproducing the emitting states. The low yield limited experiments to the region close to the gratings’blazewavelengths. It has not yet been possible to study lower energies around the ionisation threshold. The decays shown in fip. 1 are clearly non-exponential. Detailed analysis of the curves is made difficult by the “wraparound” from the preceding synchrotron pulse, shown at left in fig. 1. The intrinsic fluorescence lifetimes of DNA are very short ( < 1 ns ) [ 6 1. It followsthat the decay processesobserved in the VUV are those of the precursors of the excited states - radical ions of, possibly, triplet states [2]. At low energiesthe field effect rises smoothly with time and then levelsoff (figs. 2 and 3). This is characteristic of the geminate recombination of radical ions: hype&e interaction with magnetic nuclei makes the spin wavefunction of the pair oscillatebe-
measurement), (b) ditto (after 7 h irradiation at various wavelengths), ( c) 25 eV photons (6 h ) . Zeromagnetic field.
04 x
x
0.3
y
x
AI
xx
0
0
I 0
0.2 0
D-1 0
04 0 04
I
0
50
100
150
Fii 2. Fractional enhancement of luminescence by a field of 0.135 T: x, 15 eV, 0,25 eV. Luminescence maximum at I= 0. Channels groupedto give at least 1OOCHJ counts beforetaking ratios.
Linesrepresentlargenumbersof points:detailin Eg. 3.
130
*
Fig, 3. As for f@_2: changeswith photonenergyof the field effect
at shorttimes.
Volume 173, number 1
CHEMICAL PHYSICS LETTERS
tween singlet and triplet states [ 11. VUV has been used to demonstrate this behaviour with an alkane scintillator solution [ 31, but its occurrence in DNA is surprising for the reasons given above. At higher photon energies, the field effect changes (figs. 2 and 3 ): there is an enhancement at very short times. Such effects in crystalline anthracene [ 21 have been ascribed to fission of highly excited singlets into pairs of triplets (either two molecular triplets or one such and a triplet ion pair initially) in competition with internal conversion to the emitting state. The subsequent decrease is probably due to a reduction in the reverse process of triplet-triplet annihilation. We have found similar short-time field effects in sodium salicylate and an aromatic liquid (Santovac oil: a poly-phenoxy compound) but not in squalane (an alkane) [3]. The decrease in the field effect at longer times is probably due to initial production of triplet excited molecules along with the ion pair: the latter must then be triplet to maintain the overall singlet character of the spur. We observe similar behaviour in the other aromatic systems and the effect is so large in anthracene as to produce a negative hyperline effect [ 21. In squalane, where solvent triplets are relatively high in energy, a reduction in the field effect only occurs at higher energies (around 25-30 eV ) , where it probably results from cross-recombination of two ion pairs [3]. The changeover in behaviour between 15 and 20 eV is much more marked in DNA than in the other compounds we have studied. This is paralleled by a change in pulse shape (fig. 1): again there is a rapid change up to 20 eV, after which the shape remains approximately constant; squalane [ 31, sodium salicylate and Santovac oil show an increase in the relative intensity of the “tail” with increasing energy: DNA shows the opposite, suggesting that simple ionisation is largely replaced by another process - at least as a source of luminescence. The high-energy field effect appears to have a rise time of l-l .5 ns (fig. 3; sodium salicylate behaves similarly). This is not easily explained since the internal conversion processes competing with excited-
28 September 1990
state fission should be much faster than this. In the near UV [ 61, several decay processes are observed: it may be that only the precursors of the slower of these are affected by the field.
4. Conclusion The observed field effects have been interpreted in terms of recombination of radical ion and fission of highly excited states into triplets, the former dominant around 12- 15 eV, the latter above 20 eV. During radiolysis in vivo, the presence of water is likely to suppress recombination of radical ions. However, fission into triplets may well be a major process in radiolysis: the most probable energy loss for fast electrons (in water) is 22.5 eV [ 71. Since higher triplets of aromatics can sensitise aliphatic bondbreakage efficiently [ 8 3, the process should be considered as a possible source of double-strand breaks.
Acknowledgement The authors thank the SERC for providing facilities.
References [ 1 ] B. Bmcklehurst, Intern. Rev. Phys. Chem. 4 (1985) 279. [2] J. Rlein, J. Chim. Phys. 80 (1983) 627; P. Martin, J. Klein and R. Voltz, Physica Scripta 35 (1987) 575. [3] G.J. Baker, B. Brocklehurst, M. Hayes,A. Hopkirk,D.M.P. Holland, LH. Munro and D.A. Shaw, Chem. Phys. Letters 161 (1989) 327. [41 B. Brocldehurst, A. Hopkirk and LH. Mum-o, Radiat. Phys. Chem., in press. [ 51B. Brocklehurst, Chem. Britain 23 (1987) 53. [6] J.P. Bnllini, P. Vigny and M. Daniels, Biophys. Chem. 18 (1983) 61; S. Georghiu, T.M. Nordlund and A.M. Saim, Photochem. Photobiol. 41 (1985) 209. [ 71 S.M. Pimblott, J.A. LaVeme, A. Mozumder andN.J.B. Green, J. Phys. Chem. 94 (1990) 988. [S] B.BrockJehurst,W.A.Gibbons,F.T.Lang,G. PorterandM.1. Savadatti, Trans. Faraday Sot. 62 ( 1966) 1793.
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Volume173,number1
Luminescence of DNA excited in the vacuum ultraviolet B. Brocklehurst a, A. Hopkirkb and I.H. Munro b ’ Chemistry Department, Universityof Smeld, Sh@eld S3 7Hl? UK b SERC, Daresbury L&oratory,
Warrington WA44AD, UK
Received 1 June 1990
Pulsed vacuum ultravioletradiation has been used to exciteDNAin the energyrange12to 30eV.Time-resolved single-photon countingshowsnon-exponential luminescence decays over some hundreds of nanoseconds: the application of a magnetic field demonstrates the roleof radical-ionrecombinationand,at higherenergies,of fissionintotwotripletspecies.
1. Introduction The effects of ionising radiation are complicated by the size distribution of the spurs - groups of radicals, ion pairs, excited states, etc. The use of the vae uum ultraviolet (WV) simplifies elucidation of the primary processes: e.g., a 10-I 5 eV photon produces a single ion pair, 20-40 eV two ion pairs or an ion pair and an excited molecule. Time-resolved luminescence and magnetic field effects are sensitive probes of subsequent processes [ 11. Most previous work of this type [l-3] has concentrated on hydrocarbons because the radical ions produced initially do not undergo further reactions before recombination: in compounds of oxygen or nitrogen, ion-molecule reactions and dissociative at&hment usually produce neutral radicals and recombination does not lead to excited states. However, we now report that dry DNA irradiated in the WV Iuminesces on a time scale much longer than that of near UV excitation and that field effects can be observed.
2. Experimental The apparatus has been described previously [ 3,4]. When operated in single-buch mode, the Synchrotron Radiation Source at Daresbury provides 200 ps pulses every 320 ns with a continuous spectrum [ 5 1. A Seya monochromator (station 3.1) with two in0009-2614/90/$03.50
terchangeable gratings was used, giving intensities of the order of 10s-log photons per second at the sample. Time-resolved single-photon counting was used to measure the luminescence decays. Data were collected in separate sections of an MCA over several cycles in which an applied field (0.135 T) alternated with zero field for periods of 100 s. Salmon testes DNA (Sigma) was used as received. The results shown are from a single run with the dry material; similar results have been obtained with DNA plated from aqueous solution. Since no windows could be used, the samples were pumped down to c 100~ Torr over a period of at least an hour, and kept in vacua over,several hours during the experiment. Significant water loss from the surface layers probably occurred during this period, leading to a contraction of the helix. During the course of the experiment the pulse-shape changed (fig. 1): the field effect showed smaller changes but the qualitative results were not affected, the results in fig. 3 were obtained by alternating between higher and lower energies. Drying or radiation damage may be responsible for the changes. However, the same striking change in the field effect with energy was observed both early and late in the run.
3. Results and discussion Comparison with crystalline sodium salicylate gave a very rough estimate of 0.003 for the luminescence
0 1990 - Elsevier Science Publishers B.V. (North-Holland)
129
Volume 173, number 1
CHEMICAL PHYSICS LETTERS
l-00
O*lO
0.01
0
50
100
150 t/ns
Fig. 1.Norma&xl luminescencedecay curves for dry DNA measuredduring a single run; (a) excited by 15 eV photons (first
28 September 1990
yield in the VUV; since yields in the near UV are also low [ 61, the inefficiency is in the final luminescence step rather than in the processesproducing the emitting states. The low yield limited experiments to the region close to the gratings’blazewavelengths. It has not yet been possible to study lower energies around the ionisation threshold. The decays shown in fip. 1 are clearly non-exponential. Detailed analysis of the curves is made difficult by the “wraparound” from the preceding synchrotron pulse, shown at left in fig. 1. The intrinsic fluorescence lifetimes of DNA are very short ( < 1 ns ) [ 6 1. It followsthat the decay processesobserved in the VUV are those of the precursors of the excited states - radical ions of, possibly, triplet states [2]. At low energiesthe field effect rises smoothly with time and then levelsoff (figs. 2 and 3). This is characteristic of the geminate recombination of radical ions: hype&e interaction with magnetic nuclei makes the spin wavefunction of the pair oscillatebe-
measurement), (b) ditto (after 7 h irradiation at various wavelengths), ( c) 25 eV photons (6 h ) . Zeromagnetic field.
04 x
x
0.3
y
x
AI
xx
0
0
I 0
0.2 0
D-1 0
04 0 04
I
0
50
100
150
Fii 2. Fractional enhancement of luminescence by a field of 0.135 T: x, 15 eV, 0,25 eV. Luminescence maximum at I= 0. Channels groupedto give at least 1OOCHJ counts beforetaking ratios.
Linesrepresentlargenumbersof points:detailin Eg. 3.
130
*
Fig, 3. As for f@_2: changeswith photonenergyof the field effect
at shorttimes.
Volume 173, number 1
CHEMICAL PHYSICS LETTERS
tween singlet and triplet states [ 11. VUV has been used to demonstrate this behaviour with an alkane scintillator solution [ 31, but its occurrence in DNA is surprising for the reasons given above. At higher photon energies, the field effect changes (figs. 2 and 3 ): there is an enhancement at very short times. Such effects in crystalline anthracene [ 21 have been ascribed to fission of highly excited singlets into pairs of triplets (either two molecular triplets or one such and a triplet ion pair initially) in competition with internal conversion to the emitting state. The subsequent decrease is probably due to a reduction in the reverse process of triplet-triplet annihilation. We have found similar short-time field effects in sodium salicylate and an aromatic liquid (Santovac oil: a poly-phenoxy compound) but not in squalane (an alkane) [3]. The decrease in the field effect at longer times is probably due to initial production of triplet excited molecules along with the ion pair: the latter must then be triplet to maintain the overall singlet character of the spur. We observe similar behaviour in the other aromatic systems and the effect is so large in anthracene as to produce a negative hyperline effect [ 21. In squalane, where solvent triplets are relatively high in energy, a reduction in the field effect only occurs at higher energies (around 25-30 eV ) , where it probably results from cross-recombination of two ion pairs [3]. The changeover in behaviour between 15 and 20 eV is much more marked in DNA than in the other compounds we have studied. This is paralleled by a change in pulse shape (fig. 1): again there is a rapid change up to 20 eV, after which the shape remains approximately constant; squalane [ 31, sodium salicylate and Santovac oil show an increase in the relative intensity of the “tail” with increasing energy: DNA shows the opposite, suggesting that simple ionisation is largely replaced by another process - at least as a source of luminescence. The high-energy field effect appears to have a rise time of l-l .5 ns (fig. 3; sodium salicylate behaves similarly). This is not easily explained since the internal conversion processes competing with excited-
28 September 1990
state fission should be much faster than this. In the near UV [ 61, several decay processes are observed: it may be that only the precursors of the slower of these are affected by the field.
4. Conclusion The observed field effects have been interpreted in terms of recombination of radical ion and fission of highly excited states into triplets, the former dominant around 12- 15 eV, the latter above 20 eV. During radiolysis in vivo, the presence of water is likely to suppress recombination of radical ions. However, fission into triplets may well be a major process in radiolysis: the most probable energy loss for fast electrons (in water) is 22.5 eV [ 71. Since higher triplets of aromatics can sensitise aliphatic bondbreakage efficiently [ 8 3, the process should be considered as a possible source of double-strand breaks.
Acknowledgement The authors thank the SERC for providing facilities.
References [ 1 ] B. Bmcklehurst, Intern. Rev. Phys. Chem. 4 (1985) 279. [2] J. Rlein, J. Chim. Phys. 80 (1983) 627; P. Martin, J. Klein and R. Voltz, Physica Scripta 35 (1987) 575. [3] G.J. Baker, B. Brocklehurst, M. Hayes,A. Hopkirk,D.M.P. Holland, LH. Munro and D.A. Shaw, Chem. Phys. Letters 161 (1989) 327. [41 B. Brocldehurst, A. Hopkirk and LH. Mum-o, Radiat. Phys. Chem., in press. [ 51B. Brocklehurst, Chem. Britain 23 (1987) 53. [6] J.P. Bnllini, P. Vigny and M. Daniels, Biophys. Chem. 18 (1983) 61; S. Georghiu, T.M. Nordlund and A.M. Saim, Photochem. Photobiol. 41 (1985) 209. [ 71 S.M. Pimblott, J.A. LaVeme, A. Mozumder andN.J.B. Green, J. Phys. Chem. 94 (1990) 988. [S] B.BrockJehurst,W.A.Gibbons,F.T.Lang,G. PorterandM.1. Savadatti, Trans. Faraday Sot. 62 ( 1966) 1793.
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