3409
J. Phys. Chem. 1991, 95,3409-3415 and much less hydrated than OH-M. This environmental specificity of la involves a significant effect on its outstanding catalytic efficiency in the reaction medium. The extent of both processes, promoted by l a or OH-M is strictly related to the deprotonation degree. Although in the studied dehydrobrominations the mm value is less than 0.2, the reaction with la is predominant because the kMDvalues are approximately 10-fold higher than the values of kM. In extreme conditions of a complete ionization of the nucleophile, Le., mm z I , the reaction with OH-M can be eliminated from the kinetic considerations and the kinetic treatment becomes considerably simplified. Acknowledgment. Support of this work by the Polish Academy of Sciences, Grand RPBP 01.10, is gratefully acknowledged. The author is greatly indebted to Professor Bogdan Burczyk for his encouragement throughout this work and Professor Marian Kochman for helpful discussions, as well as Tadeusz Wilk for his assistance in computational procedure and for contributing to the Appendix.
Appendix Since the derivation of eqs 1-8 is rather complex and there are many parameters, it is only possible to obtain a solution by means of numerical calculations. Apart from the main kinetic equation (5) the following expressions are important for computational purposes:
Under the following assumptions mDOH
< 1;
a?> 1;
[B~T2 ] [Dn]
a numerical convergent algorithm can be constructed, allowing one to find a unique set of values of mmH and m&: Step 0: i = 0. Step 1: mmH,i = 1. Step 2: The value m#H,i is calculated from eq A4. Step 3: The value mOHj from the step 2 is substituted in eq A3 in order to calculate n t m ~ , ~ + ~ . Step 4: The value m$H,,+I is calculated from eq A4 by using the mDOH,i+lvalue. Step 5: If (mmH,i - mmH,i+i) < e and (dH,i+I - &,i) < C Y where e is the assumed accuracy for the best fit, then the final values are mmH = mmH,i+Iand mgH = m$HJ+I,and the calculation is terminated; otherwise, i = i + 1 and one returns to the step 3.
Relative Abundances of Primary Ion Radicals in y-Irradiated DNA: Cytosine vs Thymine Anions and Guanine vs Adenine Cations Michael D. Sevilla,* David Becker, Mengyao Yan, and Steven R. Summerfield Department of Chemistry, Oakland University, Rochester, Michigan 48309 (Received: October I, 1990)
An ESR study of the relative distribution of ion radicals formed in DNA equilibrated with D20 and y-irradiated at 77 K is presented. The ESR spectra of irradiated DNA and polynucleotides (poly[dG].poly[dC] and poly[dAdT]-polyldAdT]) were obtained and employed in a computer-assisted analysis for the individual ion-radical distribution. Analysis of spectra as a function of power allowed the separation of the spectra of the pyrimidine anions (T', C)' from the spectra of the purine The spectra of the mononucleotide ion radicals, dCMP-, dTMP-, dGMP+, and dAMP'+, were produced cations (Go+,k+). in 8 M LiCl glasses. In addition, the spectra of the ion radicals of all of the mononucleotide ion radicals except dAMP" were simulated by using hyperfine and g tensors from the literature. Basis spectra derived from (1) power saturation experiments, (2) polynucleotide and mononucleotidespectra, (3) spectra of mononucleotides alone, and (4) anisotropic simulations were used to fit the spectra of DNA by use of a linear least-squares analysis. Each of the four separate analyses confirms that the cytosine anion dominates the spectra of DNA at 100 K. Three analyses include the cationic composition. and they strongly favor the guanine cation over the adenine cation. An average of our results gives the DNA ion radicals' relative abundances as ca. 77% C', 23% T' for the anions and >90% Go+for the cations; about equal amounts of anions and cations are present. No difference in results is found for DNA irradiated in frozen D20solutions or simply exchanged at 100%D20 humidity.
Introduction The p i m a y effect of direct radiation damage to duplex DNA is the formation of cationic and anionic radicals.'-' In DNA (1) Grislund, A.; Ehrenberg, A.; Rupprecht, A.; Stram, G. Biochim. Biophys. Aria 1971, 254, 172.
0022-3654/91/2095-3409$02.50/0
irradiated at low temperature, several lines of investigation have suggested that the hole (positive charge) migrates to and stabilizes (2) Graslund, A,; Ehrenberg, A.; Rupprecht, A.; Stram, G.; Crespi, H. Inl. J . Radial. Biol. 1975, 28, 313. (3) Grgslund, A,: Ehrenberg, A.; Rupprecht, A. Int. J. Radial. Biol. 1977, 31, 145.
0 1991 American Chemical Society
3410 The Journal of Physical Chemistry, Vol. 95, No. 8, I991
on guanine.'~~Some of the same investigations have also concluded that the electron migrates to and stabilizes on thymine,'" but this conclusion has recently been called into q u e ~ t i o n . ~ Early work by Graslund et al. with oriented DNA samples suggested the thymine anion, T'-, and guanine cation, Go+,as the favored primary ionic sites, although the cytosine anion, C-, and cation, c'+, were also considered possibilities.' Later work resulted in the report that only Go+ and T'-were In photoionized DNA in an aqueous glass, Sevilla and co-workers have shown that G'+ dominates the ESR signaL8 Gregoli and coworkers concluded that, in costacked nucleotide complexes, cytosine (in preference to thymine) is the site of electron attachmentlo but that, in DNA, thymine is the anionic sitee5 Since the ESR spectra of the two pyrimidine anions To- and C'- are difficult to distinguish in model ~ystems'J*'~ as well as in DNA,' the argument regarding the identity of the anionic primary radical has been largely based on the formation of secondary radicals from the anion. The conversion of substantial amounts of the DNA anion to TH', the 5,6-dihydrothymin-5-yl radical, and the lack of any recognizable formation of cytosine secondary radical^^,'*'^ have weighed heavily in favor of T'-as the anionic primary radical. In apparent contradiction, Bernhard, using X-irradiation of oligonucleotides in LiCl glasses, has concluded that at 10 K approximately 80% of the anionic sites reside on cytosine, and he has suggested that the situation may be similar for DNA.9 In addition, Huttermann and c o - ~ o r k e r shave ' ~ recently shown, from an analysis of oriented DNA with thymine deuterated at the 5-methyl group, that the assignment of the DNA anion to T'- in earlier oriented DNA work was based on an incorrect interpretation of the ESR spectra. Consequently, other species are possible. In earlier work with dinucleoside phosphates in 12 M LiCl at 110 K, Sevilla et al. have suggested that the electron affinities of cytosine and thymine are approximately the same,'?' which also leads to the expectation that both anions should be found in irradiated DNA. This work reports an investigation of the y-irradiation, at low temperatures, of DNA and of the DNA-like polynucleotides, poly(deoxyadeny1ic-thymidylic acid) (poly[dAdT].poly[dAdT] ), poly(deoxyguany1ic)-poly(deoxycytidy1ic acid) (poly[dG].poly[dC]), and poly(guany1ic acid)-poly(cytidy1ic acid) (poly[G]* poly[C]). Through the use of a computer-assisted analysis of the composite ESR spectra of y-irradiated polynucleotides, we have generated representative ESR spectra of the thymine anion and the cytosine anion. In polynucleotide samples equilibrated with D 2 0 , the spectra of the two anions are clearly distinguishable. We have produced the individual DNA nucleotide anions and cations in aqueous glasses. Anisotropic computer simulations of nucleotide anions and cations have also been produced. Analysis of the spectra obtained from irradiated DNA in D 2 0 indicates that, at 100 K, the primary anionic site in y-irradiated DNA is a mixture of mainly the cytosine anion (ca. 77%) with smaller amounts of the thymine anion (ca. 23%), whereas the cationic site is largely guanine (>90%). Experimental Section
Materials and Methods. Salmon testes DNA (type 111, from (4) HQttermann. J.; Voit, K.; Oloff, H.; Kohnlein, W.; GrBslund, A.; Rupprecht. A. Faraday Discuss. Chem. SOC.1984, No. 78, 135. ( 5 ) Gregoli, S.; Olast, M.: Bertinchamps, A. Radiar. Res. 1982,89, 238. (6) Boon, P. J.; Cullis, P. M.: Symons, M. C. R. J . Chem. SOC.,Perkin Trans. 2 1984, 1393. (7) Sevilla, M. D. In Excited Stares in Organic Chemistry and Biochemisrry; Pullman, B., Goldblum, N., Eds.; D. Reidel: Dordrecht, Holland, 1977. (8) Sevilla, M. D.; DArcy, J . B.; Morehouse, K. W.; Englehardt, M. L. Phorochem. Phorobiol. 1918, 28, 37. (9) Bernhard, W. A. J . Phys. Chem. 1989, 93, 2187. (10) Gregoli, S.: Olast, M.; Bertinchamps, A. Radiar. Res. 1979, 77,417. (11) Sevilla, M. D.; Failor, R.; Clark, C.; Holroyd, R. A.; Pettei, M. J . Phys. Chem. 1916,80, 353. (12) Cullis, P. M.; Podmore, 1.; Lawson, M.; Symons, M. C. R.; Dalgarno, B.; McClymont, J . J . Chem. SOC.,Chem. Commun. 1989, 1003. ( I 3) Symons, M. C. R. Inr. J . Radiar. Biol. 1990, 58, 93. (14) Zell, I.; HQttermann, J.; Grislund, A,; Rupprccht, A.; Kohnlein, W. Free Radical Res. Commun. 1989, 6. 105.
Sevilla et al. salmon testes), poly [dAdT].poly [dAdT] ( MW 100OOO-500OOO), poly[dG]-poly[dC] (high MW), poly[G]-poly[C], and DNA base nucleotides were all obtained from Sigma. All samples were free of obvious contaminants except for poly[dG]-poly[dC], which contained sufficient chloride ion to be observed as C12'- in irradiated samples. Since the CI2'- signal is easily subtracted, the sample of poly[dG].poly[dC] was used without further purification. Frozen anoxic samples of D N A and polynucleotides in D 2 0 were prepared by use of vacuum techniques. The DNA or polynucleotide was vacuum-dried to remove the water of hydration, and D 2 0 was distilled onto the sample; usually the mass of D 2 0 used was at least 10 times the mass of sample. The sample was allowed to equilibrate with the D 2 0 and was then frozen in liquid nitrogen to form an ice plug. After freezing, the sample was removed from its glass tube and irradiated for doses between 0.4 and 2 Mrad at 77 K. DNA ( D 2 0 ) samples were also prepared by a second technique in which DNA was equilibrated with D 2 0 vapor at 100% humidity for 1 week under N2, and then a sample was prepared by rolling it into a 4 mm X 15 mm cylinder under N2. DNA irradiated as an ice plug or as a hydrated (D20) solid gave similar results after annealing for 1 min to 120-1 30 K to remove the ice radical signal(s). The 2'-deoxycytidine 5'-monophosphate (dCMP) and thymidine 5'-monophosphate (TMP) anions were prepared in 8 M LiCl glasses by y-irradiation at 77 K; nucleotide concentrations of 5 mg/mL were used. Irradiation produces an electron and CI2'-. The electron attaches to the nucleotide to form the anion; the C12' ESR signal is easily subtracted from the spectrum by computer subtraction lechniques. We also produced identical spectra of the anions, albeit at lower intensity, by photoionization of 8 M LiCl glasses containing K4Fe(CN), (8 mg/mL). The electrons formed by photoxidation of the ferrocyanide anion add to the deoxynucleotide monophosphate (0.5 mg/mL). The 2'-deoxyguanosine 5'-monophosphate (dGMP) and 2'-deoxyadenosine 5'-monophosphate (dAMP) cations were prepared by irradiation of 8 M LiCl glasses containing the electron scavenger, K3Fe(CN), (8 mg/mL), and nucleotide (3 mg/mL) at 77 K. Annealing to ca. 140 K allows (21;- to attack and oxidize the nucleotide. These techniques have been discussed in more detail in previous ~ o r k . ~ , ' ~All , ' ~experimental spectra were taken on a Varian Century Series EPR spectrometer operating at X-band with a dual cavity and a 200-mW klystron. Spectra were taken with Fremy's salt ( g = 2.0056, AN = 13.09 G) as a reference. Computer Analyses. Each spectrum was stored in a 1000-point array with field calibration marks from the three Fremy's salt ESR lines. DNA and polynucleotide samples resulted in composite spectra, with both anion and cation primary radicals present. Individual ion-radical spectra were used to analyze the composite DNA spectra for ion-radical composition. The three experimental methods employed to generate the spectra of individual ion radicals are briefly described below. 1 . Power-Differentiated Spectra. In nucleotides, polynucleotides, and DNA, the ESR resonances of the C'- and Toradicals were more easily power-saturated than those of A'+ and Go+. Thus, it was possible to separate the anion spectrum from the cation spectrum by subtraction of spectra at different powers." Since there was some ambiguity regarding how much of one spectrum to subtract from another, the following criteria were used. The cation to anion ratios present in the irradiated samples were assumed to be 1/ 1 unless hole scavengers such as CI-were present. In samples in which CI- scavenged holes, we observed a higher anionlcation ratio. Since the cations are singlets and the anions doublets, in the subtractions we had only to decide on (15) Sevilla, M. D.; Swarts, S. J. Phys. Chem. 1982, 86, 1751. (16) Sevilla, M. D.: McGlashen, M. J . Phys. Chem. 1983.87, 634. (17) Power saturation analyses were performed on individual nuclootides in 8 M LiCl glasses as well as on irradiated DNA, poly[G1.poly[C]. and poly[dAdT].poly[dAdT]. The individual nucleotide cation radicals (dGMP+, dAMP'*) and anion radicals (dCMP-, T M P ) exhibit similar saturation behavior, as is found for the resptctive cationic and anionic components in the polynucleotides (Figure 3), except that the anions show a constant intensity at higher powers (Sevilla, M. D.; Becker, D.; Summerfield, S. Unpublished results).
Primary Ion Radicals in y-Irradiated DNA
Figure 1. First-derivative ESR spectra of 77 K y-irradiated and D20-
equilibrated DNA and polynucleotide samples. Samples were warmed to 130 K and spectra recorded at 100 K: (A) Salmon testes DNA equilibrated with D20 at 100%humidity; (B) poly[G]-poly[C] in DzO; (C) poly[dG].poly[dC] in DzO (D) poly[dAdT].poly[dAdT] in D20. All spbctra were recorded at low powers (ca.45 dB, 6.3 pW). The markers are separated by 13.09 G with the central marker at g = 2.0056. The magnetic field increases from left to right in all figures. the depth of the 'switchback" in the anion doublet, consistent with the maintenance of a singlet line shape for the cation. The combined criteria resulted in an uncertainty of about 5% in the subtracted amount. 2. Individual Ion Radicals. The spectra of the ion radicals (C*-, T-, Go+,A'+) were obtained for individual nucleotides in LiCl. 3. Anion Spectra from Polynucleotides. The spectra of C'and T'-were also generated by subtraction of the spectrum of dGMP'+ (dAMP'+) from the spectrum of poly[dG].poly[dC] (poly[dAdT].poly[dAdT]).The anion/cation ratio was assumed to be 1 / 1 for poly[dAdT] and, owing to the presence of C1-, 0.65/0.35 for poly[dG].poly[dC]. The spectra of C'-, T*-, Go+,and A'+ generated as described above were employed to analyze DNA for its ionic radical composition. In this method, individual spectra (basis spectra) that make up the composite spectrum were summed and the result was compared with the experimental spectrum. The complete set of spectra needed to reproduce a composite spectrum was called a basis set. Here we assumed the spectra of Go+,A.+,C', and T'formed such a set.Is A least-squares error function (ER) was generated as the sum of the squares of the differences in the heights of the simulated spectrum and the experimental spectrum: ER =
X[yexp(4- Y~i,(i)12/C[y~,p(i)12
A recursive computer program generated the fit that gave the smallest value of ER. Checks were routinely performed to ensure that the spectral sum did not converge on a local minimum in ER and also to ensure that the order of summing of the individual basis spectra had no effect on the final results. In our table we report the residual error function, R, as defined by Bernharde9 We find it tracks closely with (ER)'12. The wings of the spectra, the first 300 points and the last 250 points, were not used in the fitting, as they were mainly noise and base line. In comparing our spectra, note that the first field marker is at point 320, the last at 620, and they are separated by 26.18 G. Fits that covered a larger range, i.e., from point 200 to point 850 gave very similar results; they differed by no more than 3% in any basis function, but the error function, R , was approximately 20% larger. (18) Our techniques are similar to those employed in our previous work7*" and by &mhard9 on oligonucleotideanions in LiCl glasss His work showed no evidence for the presence of G' and a minor perhaps negligible fraction of A'. From our previous work with pyrimidine cations,' we can eliminate any but very minor amounts of T'+.
The Journal of Physical Chemistry, Vol. 95, NO. 8, 1991 3411
Figure 2. Power saturation behavior of the DNA sample of Figure 1A at 100 K. The microwave power levels range from 0.63 pW, 55 dB (A) to 6.3 mW, 15 dB (E). The spectrum of the anion is found to be. power-saturated more readily than that of the cation; as a consequence, the cation spectrum dominates the higher power spectra.
Results In Figure 1, we present the ESR spectra for DNA (A), poly[GI.poly[Cl (B), poly[dGI.poly[dCl (C), and pol~[dAdTl-poly[dAdT] (D) y-irradiated at 77 K and annealed to 130 K to remove any ESR signal from the ice component of the sample. In accord with previous work, we assume the spectra arise predominantly from the purine cations (G'+,A.+) and pyrimidine anions (TO-, C'-). The resolution observed in the spectra arises largely from the anionic components because the purine cations are poorly resolved singlets in polycrystalline media.8 The DNA, poly[dG].poly[dC], and poly[G].poly[C] spectra are remarkably similar. A cursory comparison of the spectra indicates that the cytosine anion must make a significant contribution to the spectrum in DNA. In sections below, the results of methods used to quantify the contributions of the individual ion radicals are discussed. Differential Power Saturation. In Figure 2, we present the effect of power saturation on the shapes of the spectra of DNA at 100 K, and in Figure 3A a plot of the spectral intensity vs (power)'/2 is given. Power saturation does not occur a t power levels below 6.3 pW; i.e., at powers up to 6.3 pW (45 dB), spectral intensities are proportional to the square root of the power. With increasing power, the anion-radical signal is saturated more readily than that of the cation. Its spectrum progressively becomes a smaller fraction of the signal; this is evidenced by the disappearance of the anion doublet signal as the power is increased. The anion signal also broadens with power, but the doublet remains resolved until about 20 dB. As evidence for this, notice the resolution of the second peak in the low-power spectrum of DNA (Figure 2A). This resolution is still observed although the peak is less resolved even a t 25 dB; at this power, since the anion contributes only about 25% of the signal, its spectrum is spread over the more intense cation singlet. Since the anion signal is saturated more easily than that of the cation, subtraction of the 25-dB spectra from the 45-dB spectra in the correct proportion gives the anion signal only. From such subtractions a t various powers, a power saturation curve for the DNA anion and cation components in DNA was obtained (Figure 3A). The ion radicals of poly[G].poly[C] and poly[dAdT].poly[dAdT] were found to have power saturation behavior similar to that found in DNA (Figure 3B,C). The falloff in the intensity of the anions at higher powers is likely an artifact of the analysis, since no correction for broadening of the spectra with power was made.17 It is of interest to note that to this date it is likely that spectra reported for the anion radicals of pyrimidine DNA bases were often power-saturated to some Figures 4A, 5A, and 6 show the power-differentiated anion spectra for C'- in poly[dG].poly[dC], T'-in poly[dAdT]-poly[dAdT], and DNA'- in DNA, respectively. It is clear from these
Sevilla et al.
3412 The Journal of Physical Chemistry, Vol. 95, No. 8, 1991
V Figure 4. ESR spectra found for C- from four different methods: (A) differential power saturation analysis of poly[dG].poly[dC] at 100 K (B)
computer simulation from hyperfineand g tensors from literature values (see text); (C) spectra of the dCMP anion formed by electron attachment to dCMP in 8 M LiCl at 100 K (D) spectrum found by computer subtraction of the dGMP'+ spectrum from that of poly[dG].poly[dC]. These spectra are similar in line shape, resolution, and doublet separation; the only exceptions are that (A) shows slightly more resolution of the upfield peak and (C) is somewhat broadened throughout.
0.0
0.5
1 .o
P(mW)"1/2 Figure 3. (A) Plot of the total radical intensity found by double integration of the spectra of irradiated DNA of Figure 2 as a function of (power)'/*. The curves found for the DNA anion (DNA'-) and DNA cation (DNA'+) are found by the analysis of the DNA using spectra isolated by differential power saturation. (B, C) Same analysis as in (A) for (9) irradiated poly[G].poly[C] in D20 and (C) irradiated poly[dAdT].poly[dAdT] in D20. All spectra used in the analyses were recorded at 100 K. The saturation behavior for DNA and all the polynucleotides shows a linear relation between intensities and (power)'12 up to 6.3 pW.
subtractions that the DNA anion (Figure 6) is dominated by the cytosine anion (Figure 4A). The thymine anion (Figure SA) has no resolved structure on the upfield peak and clearly cannot be a major contributor to the DNA anion spectrum. In Figure 4B, we show a computer-simulated spectrum of C,' obtained by use of an anisotropic simulation19with parameters published for the proton coupling for the Y-dCMP radical anion in a single crystal (-22.6, -7.3, -1 1.8 G)20and two anisotropic nitrogen couplings (0, 0, 4 G ) and g values (2.0034, 2.0039, 2.002 1 ) estimated from those reported for oriented DNA' and for the anion of 2'-dUMP.21 The line width in all simulations was varied from 4 G in the central portion of a spectrum to 6 G in the wings (see discussion below). The simulation shows the resolution of a small component at the top of the upfield peak, as found in the spectrum of Figure 4A. In addition, we show the spectrum of C'- formed by electron attachment to dCMP in 8 (19) Lefebvre, R.;Maruani, J. J . Chem. Phys. 1965, 42, 1480. (20) Close, D. M.; Bernhard, W. A. J . Chem. Phys. 1979, 70, 10. (21) Voit, K.; HIlttermann, J. J . Magn. Reson. 1983, 55, 225.
I
1.5
I
V
Figure 5. ESR spectra found for T' from four different methods: (A)
differential power saturation analysis of poly[dAdT].poly[dAdT] at 100 K; (B) anisotropic computer simulation from hyperfine and g tensors from literature values (see text); (C) spectrum of the TMP anion formed by electron attachment to TMP in 8 M LiCl at 100 K; (D) spectrum found by computer subtraction of the dAMP'+ spectrum from that of poly[dAdT].poly[dAdT]. The spectra of T'-lack the resolution at the top of the upfield peak found for C'. In addition, the splitting between the doublet components is smaller by ca. 2 G in all spectra of T'-than that found for C'- (Figure 4). Both these points make it possible to easily distinguish the two anion spectra.
M LiCl (Figure 4C). The spectrum in the LiCl glass is less resolved but still shows indications of a small component on the upfield peak. All three C'- spectra are in reasonable agreement. In Figure SB, we show an anisotropic simulation of T'-based on parameters published for the proton coupling for the thymidine anion in a single crystal (-19.8, -4.6, -10.7 G),2u3one anisotropic nitrogen coupling (0, 0 , 4 G),"' gvalues (2.0034,2.0038,2.0022) and a 4 - 6 4 line width. The nitrogen coupling in the simulated spectra of T'-or C'- only affects the line shape in the wings and not the structure in the central region of each spectrum, which is due to the anisotropic coupling of the hydrogen at C-6. The spectrum of To-formed by electron attachment to TMP in 8 M LiCl is also shown (Figure SC). The To- spectra show good agreement. Although the top of the upfield peak shows a more flattened appearance in the simulation (Figure 5B) than in the (22) Box, H.C. Radiation Effects: ESR and ENDOR A d y S i S ; Academic Press: New York. 1977; p 156. (23) Sagtuen, E.;Hole, E. 0.; Nelson, W. H.; Close, D. M. J . Phys.
Chem. 1989, 93, 5974.
Primary Ion Radicals in y-Irradiated DNA
Figure 6. Solid line: ESR spectrum found for the anion radical of DNA (DNA-) from differential power saturation analysis of y-irradiated salmon testes DNA (D20 equilibrated) at 100 K. Dashed line: result of least-squares fitting of power-differentiated spectra, 83% C- from poly[dG].poly[dC] (Figure 4A) and 17% T- from poly[dAdT].poly[dAdT] (Figure SA), to DNA. power-differentiated spectrum (Figure SA), the overall spectrum is easily distinguished from that found for C.' Most importantly, the spectra for T'-show a smaller doublet separation than that found for C*-. From these comparisons, we conclude that the spectra shown in Figures 4A, SA, and 6 are reasonable representations of the spectra of C'- and T'-in polynucleotides and DNA'- in DNA, respectively. In Figure 6, we present the results of a least-squares fit of the DNA' spectrum with the powerdifferentiated spectra of C' from poly[dG]pdy[dC] (Figure 4A) and T'-from poly[dAdT]-poly[dApdT] (Figure SA) as the basis set. This analysis gave 83% C'- and 17% T'- with R = 0.10. The simulation shows a good fit in resolution and splitting; however, line width differences result in the larger than expected value of R. G'+ and A'+ spectra derived from differential power saturation analysis were broadened and not sufficiently distinguishable to be employed to determine the distribution of the cation component on DNA. Basis Spectra for Computer Analysis of DNA Spectra. Although the results of the above analysis seem clear-cut and are in agreement with those of Bernhard for oligonucleotides: the use of C'- and T'-spectra derived from power saturation experiments has a difficulty; i.e., an unknown degree of distortion is introduced into the spectra by the broadening that accompanies power saturation. The similarity of the power-differentiated spectra to the simulated spectra and experimental spectra in LiCl suggests the distortion is not too great. However, since the power-differentiated spectra for Go+ and A'+ were not clearly distinguishable, we chose to also analyze the composite DNA signal (anions and cations) using unsaturated (45 dB, 6.3 pW or less) and simulated ESR spectra. Three different sets of basis spectra were used for the analysis of the DNA spectrum in Figure 1A. The spectra of C'- from dCMP (Figure 4C), T'-from T M P (Figure SC), A'+ from dAMP (Figure 7A), and G'+from dGMP (Figure 7B), all in 8 M LiCl glasses, were produced as described in the Experimental Section. The most straightforward analysis is the fitting of these four basis spectra to the DNA spectrum; this set of basis spectra is labeled set 111. This set has the obvious advantage that spectral contamination of one nucleotide radical ion in the spectrum of another is eliminated. However, Bernhard and Patrzalek have pointed out that the anionic spectra in polynucleotides are slightly different from spectra of the individual nucleotide anions.*' Part of this is due to the fact that protonation at 0 - 4 in the thymine anion and N-4 in the cytosine anion can produce coupling to a fl proton (CO-H or CN-H) whose orientation, and consequently coupling, depends on its environment. Since the hyperfine coupling of deuterium
The Journal of Physical Chemistry, Vol. 95, No. 8, 1991 3413
V
Figure 7. (A) ESR spectrum found for dAMP'+ in y-irradiated 8 M LiCl containing Fe(CN)6p as an electron scavenger. (B) ESR spectrum found for dGMP'+ in y-irradiated 8 M LiCl containing Fe(CN)6h as an electron scavenger. The dAMP and dGMP cations are formed by oneelectron oxidation of the nucleotide (3 mg/mL) by CI2'. (C) Anisotropic computer simulation from hyperfine and g tensors from literature values for G" in DNA.
is 1/6,5ththat of hydrogen, our use of deuterated samples avoids this problem to a large extent. Other environmental effects are still expected, but the effect of the environment is expected to be somewhat less on the cation spectrum than on the anion spectrum, due to the fact that the cation spectrum is initially less resolved. In Figure 7C, we present an anisotropic computer simulation of the DNA guanine cation spectrum based on parameters found by Huttermann et al! in oriented DNA (one anisotropic hydrogen coupling (-4.0, -8.6, -6.8 G),two nitrogen couplings (0,0, 6.8 G and 0, 0, 13.8 G), and g values (2.0043, 2.0038, 2.0021) and a variable line width of 4-6 G, as described below. The simulated spectrum is very similar to the experimental spectrum of G'+from dGMP in LiCl (Figure 7B). Anisotropic simulations based on spectra of the guanine cation in guanine single crystals fit slightly less well.25 The similarity between the simulated and the experimental spectra of Go+suggests that subtraction of the experimental nucleotide cation spectrum of d G M P + (dAMP+) from the spectrum of the polynucleotide, poly[dG].poly[dC] (poly[dAdT]q~~ly[dAdT]), should provide reasonable anion basis spectra. The resulting spectra are shown in Figure 4D for C'- and in Figure SD for T'-.This set of spectra, Go+ from dGMP (Figure 7B), A'+ from dAMP (Figure 7A), C'- (Figure 4D), and T'-(Figure SD), is called basis set I. The final basis set (11) is composed of spectra derived from the anisotropic computer simulations of C', T,' and Go+spectra only. These simulations are presented in Figures 4C, SC, and 7C. As was mentioned above, the line width was varied in these simulations. It is usually the case when nitrogen couplings are involved that the gll region found in the wings of the spectra has a greater linewidth than the g, region in the center. For this reason the line width was varied from 4 G in the central region of the spectrum (bounded by the maximum and the minimum heights) to 6 G in the wings of all three simulations. A description of the basis sets is given in Table I. Computer Analysis of DNA Spectra. The three basis sets were fit to the experimental spectra for irradiated DNA (exchanged with D 2 0 a t 100% humidity) by using the least-squares fitting procedure described earlier; the results are detailed in Table I. The fit of basis set I is superimposed on the DNA (DzO) spectrum in Figure 8A and is very good throughout the entire spectral region. Basis set I1 produces a simulation nearly identical with that for basis set I (see superposition in Figure 8B). The error function, ~
(24) Bernhard,
W.A.; Patrzalek,
A. 2. Radial. Res. 1989, 117, 379.
~~~
~
(25) Close, D.M.; Sagstuen, E.;Nelsen, W.H.J . Chem. Phys. 1985,82,
4386.
3414 The Journal of Physical Chemistry, Vol. 95, No. 8, 1991
Sevilla et al.
TABLE I: Primarv Ion-Radical Percentwe Composition of r-Irradiated DNA at 100 K
A'+
d d
basis set' (100% humidity DzO) I* I1 Ill 42.1 41.4 46.3 16.3 11.9 12.6 38.5 (34.4)' 46.0 3.1 d (7.3)'
R
0.10
0.048
radical C'ToG'+
PS 41.5 8.5
avC 43.3 13.6 39.6 3.5
basis set4 (100 mg of DNA/mL of DzO) Ib IIb I11 avC 41.3 43.7 41.7 40.1 10.3 13.0 14.0 18.7 48.3 38.8 41.2 42.8 d 4.6 1.5 d
(h5)
0.044
0.1 1
(h5)
0.043
0.042
0.098
'Basis set PS is composed of spectra for C'- (Figure 4A) and T'-(Figure 5A) derived from power saturation experiments on irradiated poly[dG].poly[dC] and poly[dAdT].poly[dAdT], respectively. Basis set I is composed of pyrimidine anion spectra formed by subtraction of spectra of dGMP+ and dAMP+ from the polynucleotide spectra, Le., C'- (poly[dG].poly[dC]+- - 0.35dGMP") (Figure 4D) and T'-(poly[dAdT].poly[dAdT]' - OSdAMP'+) (Figure 5D) as well as the cation spectra dGMP" (Figure 7B) and dAMP'+ (Figure 7A). Basis set 11 is composed of anisotropic computer simulation spectra of C'- (Figure 4B), T'-(Figure 5B), and G" (Figure 7B) from literature values of g and A tensors. Basis set 111 is composed of the spectra of the individual nucleotide ion radicals dCMP'- (Figure 4C), dTMP'- (Figure 5C), dGMP" (Figure 7B), and dAMP'+ (Figure 7A) in 8 M LiCl (in DzO).bThe values reported for basis set I are the averages of four separate fits using g variation (&0.0002 maximum) and base-line corrections. Of all values in the individual simulations, the greatest deviation from the average was 4%. Simulations with narrower line widths (in basis set 11) were found to better fit spectra of DNA (100 mg/mL of DzO).The reported value averages the two simulations. CAverageof basis sets 1-111. dThe cation/anion ratio was assumed to be 1 / 1 for analyses based on power saturation. The PS spectra of the cations were not sufficiently distinguishable to give reliable analyses. 'Simulations with basis set 111 gave small changes in the error function for moderate changes in the G'+/A'+ ratio (Le. from 3 to 12% A"). The C'-/T'- ratio and the anion/cation ratio remained nearly constant over this range of A'+ and G'+ values. INegative values for A" resulted if the A" spectrum was included in these simulations. As a consequence, the weighting of this spectrum was held at zero. that has an ESR signal too broad to be observed has occurred. An imbalance in anion and cation was also reported by Graslund et al.2 It is important to note that our estimates of error are about f5% for each ion radical. Thus, the expected 50/50anion/cation ratio is within these limits of error.
Figure 8. (A) Result of the least-squares fitting of basis set I (dashed line) to the spectrum of y-irradiated salmon testes DNA (100% D20 humidity) at 100 K (solid line). (B) Result of the least-squares fitting of basis set I1 (dashed line) to the same DNA spectrum as in (A) (solid line). (C) Result of the least-squares fitting of basis set I (dashed line) to the spectrum of y-irradiated ice containing salmon sperm DNA (100 mg/mL D20) at 100 K (solid line). See Table I for fractional compo-
sition of the anion (C*-, T'-) and cation (Go+,A'+) components.
R, for both simulations has low values (0.048 for I; 0.044 for 11). The fit for basis set 111 is poorer in terms of the error function (0.11); however, the percentage compositions of the various ions show the same trends as is the case for I and 11. For DNA in a D 2 0 ice, similar results were found (see the superpositions in Figure 8C and Table I). Results with mixed sets of basis spectra gave similar results: for example, with the use of the simulated spectra of Go+from basis set 11,'C from basis set I, and T' from basis set JJJ, we find 43% C*-,I I % T-, and 46% Go+. The interchangeability of these basis spectra is important, since it suggests they are representative of the spectra of the primary radicals. Three points of the analyses shown in Table I are pertinent. First, the average of all three basis sets gives relative anion yields Second, all analyses suggest less of about 75% C" and 25% T-. than 10% of the cation is A'+. Work with irradiated oriented DNA has also suggested little or no A'+.4 Third, the analyses for all basis sets show a small imbalance in cation (44%)and anion (56%). This likely reflects small errors inherent in double integration of experimental ESR spectra, although it is possible that scavenging of holes by impurities or formation of a DNA radical
Conclusions Our results show that cytosine is the major site for electron attachment in DNA irradiated at low temperatures. Using anionic basis spectra derived from the subtraction of spectra at different microwave powers, our analysis of the DNA anion from DNA (D,O) indicates that, at 100 K, the anion is divided between the pyrimidine bases, with about 83% cytosine and the remainder thymine. The vagaries of differentiating spectra by power saturation stimulated us to perform further analysis using unsaturated spectra arranged into three basis sets arising from spectra of the individual DNA bases, polynucleotide spectra, and computer simulations. The results for the anion distribution using basis sets 1-111 (75% C' and 25%T-) agree within experimental error with those reported from power saturation, although the former produce far superior fits (compare Figures 6 and 8). The equal weighted average of all four results for the DNA anion give 77% (&lo%) C'- and 23% (*lo%) T+. The error limits encompass all values found from the four techniques. The average of three basis sets for the DNA cation suggests that it is principally (>90%) Go+. We find that both DNA exchanged with D 2 0 at 100% humidity and DNA frozen in a D 2 0 ice give nearly identical results. Gregoli et al. suggested that the ice radicals formed in frozen matrices are trapped in separate ice crystallites and are not able to attack DNA.S Our results show no significant differences for samples irradiated in ices or simply hydrated. They are consequently supportive of this conclusion. These results are for D20-equilibrated DNA; since it is unlikely that there would be large isotope effects on the initial ion-radical disposition, they should be equally applicable to H,O-equilibrated DNA. For example, Bernhard reports about 80% C'- anion for an oligonucleotide containing all four DNA bases in 12 M LiC1(H20) at 10 K.9 In principle, the relative abundances of the ion radicals stabilized at 77 K should depend on at three main factors, viz., the electron affinities of the bases for anions, the ionization energies of the bases for cations, and any stabilization provided by subsequent events such as proton transfer between DNA base pairs.% Sevilla et al. have suggested that for dinculeoside phosphates the electron affinities of C and T are nearly equal but are larger than for the (26) (a) Steenken, S . Chem. Rev. 1989,89,503. (b) Steenken, S.Rodiat. Res., in press.
Primary Ion Radicals in y-Irradiated DNA
The Journal of Physical Chemistry, Vol. 95, No. 8, 1991 3415
purines." Thus, the electron affinities should confine the excess electron to the pyrimidines but may not play the dominant role in determining the relative abundances of C' and T-. However, for the cations, gas-phase ionization energies (IEs) give a clearer picture. Guanine has a significantly lower IE than adenine (ca. 7 kcal), and both purines have substantially lower IEs than the pyrimidines.*' The state of protonation of DNA ion radicals has been recently discussed by Steenken.26 H e has proposed a model in which the localization of ion radicals is driven by the transfer of a hydrogen-bonded proton between complementary base pair radical ions. Equilibrium constants for such proton transfer calculated by Steenken from pulse radiolytic measurements of the individual nucleoside ion radicals in aqueous solution at room temperature suggest that C' is stabilized by the donation of a hydrogen-bonded proton from a complementary G , forming C-(H+)'. The equilibrium constants indicate that similar stabilization does not occur for T-, wen though protonation of T' at the oxygen at C-4 occurs readily in solution28 and has been shown to occur in thymidine single crystals.u This model provides a mechanism for localization of the excess electron on C in preference to T and is consistent with our low-temperature results. However, the electron affinities of C and T are not well-known; therefore, the mechanism for excess electron localization in DNA must still remain an open question. Such reversible protonation reactions could affect the results of our work if the state of protonation (deuteration) for the various basis spectra employed is not the same as for DNA. Our results for the cytosine anion in 8 M LiCl showed somewhat less resolution than was found for polynucleotides. It is possible this was due to the deuteration of the exocyclic nitrogen in C'-, which is expected in 8 M LiCl but not in p~ly[dG].poly[dC].~J~ Since it is well established that, when irradiated DNA is annealed to higher temperatures (ca. 170 K), the thymine anion
irreversibly protonates at C-6 to form the 5,6-dihydrothymin-S-y1 radical, TH', it appears that the electron originally located on cytosine migrates to thymine on annealing and is "fixed" there by protonation of the thymine anion. In all likelihood, a t elevated temperatures an equilibrium exists between C' (or C(H+)') and 7-,and irreversible protonation at thymine eventually shifts the anionic path to TH':798*1'726 C'T'- TH'
(27) McGlynn, S. P.; Dougherty, D.; Mathers, T.; Abdulner, S. In Excited Stares in Organic Chemistry a d Biochemistry; Pullman, B., Goldblum, N., Eds.; D. Reidel: Dordrecht, Holland, 1977; pp 247-256. (28) Deeble, D. J.; Das, S.; von Sonntag, C. J . Phys. Chem. 1985,89,5784.
48-4; A", 74431-04-2; dCMP'-, 72257-57-9; dTMP'-, 132696-16-3; dGMP'-, 55056-59-2; dAMP'-, 132696-17-4;poly[G].poly[C], 2528045-9; dCMP, 1032-65-1; TMP, 365-07-1; dGMP, 902-04-5; dAMP, 653-63-4.
-
Our results clearly suggest that the guanine cation is the dominant radical cation, as has been suggested from other ~ o r k . ~ * ~ ~ * The best fit to the DNA spectrum was found with a basis set that employed a spectrum of the guanine cation simulated from parameters from aligned DNA results; this fit suggests no adenine cation was present. The overall results for both DNA exchanged with D,O at 100% humidity and DNA frozen in a D 2 0 ice suggest less than 10% of the DNA cation sites are at adenine. Since DNA often has long stretches of A-T pairs, it would be surprising if the adenine cation is not present, especially since the transfer of holes in DNA bases is thought to be a less effective process than electron t r a n ~ f e r . For ~ the cation, the equilibrium model proposed by Steenken does not provide a significant stabilization energy as compared to the difference in ionization energy. Thus, the major driving force for the localization of the positive charge must be the lower ionization energy of guanine.
Acknowledgment. We thank the Office of Health and Environmental Research of the Department of Energy (Grant No. DE FG0286ER60455) and the National Cancer Institute of the National Institutes of Health (Grant No. ROlCA45424) for support of this work. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of S.R.S. as an undergraduate research student. Registry No. Poly[dG].poly[dC], 2551 2-84-9; poly[dAdT].poly[dAdT], 26966-61-0; T'-, 34504-16-0; C*-, 34480-97-2; G", 58798-
