Postirradiation Electron Transfer vs Differential Radical Decay in X

14976

J. Phys. Chem. B 2006, 110, 14976-14987

Postirradiation Electron Transfer vs Differential Radical Decay in X-Irradiated DNA and Its Mixtures with Additives. Electron Spin Resonance Spectroscopy in LiBr Glass at Low Temperatures Chandrima Pal and Ju1 rgen Hu1 ttermann* Fachrichtung Biophysik, UniVersita¨t des Saarlandes, Klinikum Bau 76, 66421 Homburg, Germany ReceiVed: December 14, 2005

Free radical formation in DNA and in colyophilized mixtures of DNA with the additives mitoxantrone and riboflavin was monitored after X-ray irradiation in frozen aqueous glasses (7 M LiBr/D2O) at 77 K by electron spin resonance (ESR) spectroscopy. Specifically, the postirradiation time course at 77 K of the respective free radical intensity residing on DNA or on the additive was probed in order to test the hypothesis of electron transfer from DNA, e.g., to mitoxantrone after irradiation under these conditions (e.g., Messer, A.; Carpenter, K.; Forzley, K.; Buchanan, J.; Yang, S.; Razskazovskii, Y.; Cai, Z.; Sevilla, M. D. J. Phys. Chem. B 2000, 104, 1128). For both additives, different additive loadings and irradiation doses were employed. The observed relative change in contributions of DNA and of additive radical components to the experimental spectra with time could be ascribed, for both additives, unequivocally to independent, differential fading of component radicals. Transfer from DNA to the additive, e.g., by electron tunneling as proposed before could be ruled out to occur by a detailed, quantitative analysis of the experimental spectra using reconstruction techniques. Additional studies were performed with the nucleotides TMP and dCMP and its mixtures with mitoxantrone in order to describe the time course in systems which are expected to behave independently; the results supported the conclusions arrived at from the analysis of the DNA/additive system. A model was proposed to describe the postirradiation radical fading mechanisms which involve liberation of radiation-induced matrixtrapped defects with time. It was assumed that these defects are ESR-mute and react with radicals by net radical destruction. Some experimental observations are presented concerning influence of temperature and of the matrix on the fading processes. These seem to argue in favor of such a model although a detailed, quantitative description is still not possible.

Introduction From the time of the discovery of the DNA structure in 1953,1 this molecule, because of its key role as a carrier of genetic information in all living organisms, has provided a vast field of research interest to scientists of diverse scope. In 1962 it was already suggested that the unique structure of B-form DNA containing double strands of π-stacked base pairs might serve as a pathway for charge migration.2 Studies on the interaction of ionizing radiation with cells have revealed that mutagenic and carcinogenic effects observed are due to damage caused in the DNA of the cell nucleus.3 These in turn were found to result from the initial formation of oxidized or reduced species on the pyrimidine, the purine, or the sugar phosphate constituents of DNA.4-8 In the past 10 years intense studies on photoactivated charge transfer in DNA9,10 containing donors (D) and acceptors (A) have emphasized a distance-independent longrange charge transfer along DNA and have invoked the molecular wire11 like model of DNA. Other investigations have brought into light a short-range charge transfer12 as well as the semiconducting13 or insulating14 properties of DNA. Since then the topic of whether DNA is a suitable medium for charge transfer has remained a subject of controversy.15,16 Theoretical models of charge transport in DNA have discussed coherent single-step electron or hole tunneling,17-19 incoherent multistep * To whom correspondence may be addressed. E-mail: bpjhue@ uniklinik-saarland.de. Tel.: ++49/6841/1626200. Fax: ++49/6841/ 1626227.

phonon assisted hopping,16,17,20 clustering,21 activation energy control,22 or soliton23 and polaron assistance.24 All these studies have led to the view that charge transfer can occur through DNA, and the subject has been moved on to topics of biotechnological relevance.25 Electron spin resonance (ESR) spectroscopy owing to its specificity in free radical detection has contributed strongly to the topic of localization of electrons and holes upon ionizing irradiation in different forms of DNA, e.g., oriented fibers, single crystals of oligonucleotides, freeze-dried amorphous samples, and in frozen aqueous solutions or glasses.14,26 A number of studies has dealt also with the influence of electron scavengers in irradiated DNA which modulate the radical balance of the constituting components in DNA.26,27 ESR spectroscopy has initially supported the idea of the mobility of electrons and holes along DNA by finding that holes are preferably trapped by guanine and that cytosine and thymine act as the main sink for electrons.6,28 Although further primary radical sites have been described in recent years,26 the total number of structures is still limited. Therefore, radical formation in DNA by ionizing radiation as observed directly after exposure does reveal a certain degree of specificity as a consequence of electron and hole mobility. We denote this phase the intrairradiation period. So far, there has been no detailed model on the transfer processes during ionizing irradiation which leads to the initial selectivity. On the other hand, several studies in recent years have applied the ESR technique to study postirradiation charge transfer. For

10.1021/jp0583086 CCC: $33.50 © 2006 American Chemical Society Published on Web 07/11/2006

Response to Ionizing Radiation in DNA electron transfer, one intensely studied system was a frozen LiBr glass consisting of DNA doped with a randomly spaced electron affinic intercalator, e.g., mitoxantrone (MX).29-36 In the glass, the holes produced in the aqueous matrix by irradiation at 77 K are scavenged by bromide ions giving rise to Br2-. This species has a broad, “baseline”-type ESR signature. Thus, the ESR spectra of the electron adducts to DNA and to the intercalator can be monitored selectively with good resolution. With this technique, the change in radicals residing on DNA and on the intercalator has been followed, at 77 K, over a large time scale after irradiation at that temperature. An increase in the ESR signal intensity of the intercalator radical compared to that of the DNA electron adducts was observed and assigned to postirradiation electron transfer from the DNA radicals to the intercalator. A superexchange tunneling mechanism was invoked giving an average value of distance dependence of electron-transfer rate, β ) 0.9 Å,32 which is temperature independent. This basic approach was subsequently applied to study interstrand vs intrastrand transfer and to elucidate the contributions of temperature-dependent processes with respect to tunneling. In our laboratory, recent studies dealt mainly with unraveling the various radical components obtained from irradiated polymeric DNA at 77 K using different levels of hydration, additives, and radiation quality26 as modulating factors and applying, e.g., high magnetic fields for disentangling overlapping spectra.37 In most of this work and specifically in thermal annealing studies done on the DNA-histone complex chromatin,38 it has been observed that after free radicals are formed and stabilized by irradiation at low temperature (intrairradiation period) they remain confined in separate compartments or deep traps. Their subsequent reactions which were so far studied by us after thermal annealing comprised mainly protonation and deprotonation. These appeared to take place within their immediate vicinity of the primary radicals and without any interaction with each other. This apparent discrepancy in the postirradiation behavior of DNA radicals with temperature and time prompted us to reconsider first the time-dependent electron transfer in irradiated DNA at 77 K. For a detailed insight, we have used DNA as well as single deoxyribonucleotides. As electron scavengers MX and riboflavin (RF) were employed which differ in their mode of binding with DNA. MX intercalates with DNA and RF forms a close association in the vicinity of guanine residues.39 Control measurements also involved a stable free radical, TEMPOL (4hydroxy-2,2,6,6-tetramethylpiperidin-N-oxyl). The chemical structures of these compounds are shown in Chart 1. Materials and Methods DNA (sodium salt from salmon testes), deoxyribonucleotides (thymidine 5′-monophosphate (sodium salt) (TMP) and 2′deoxycytidine 5′-monophosphate (sodium salt) (dCMP)), deuterium oxide (D2O), riboflavin, and mitoxantrone dihydrochloride were bought in their highest purity from Sigma (Munich) and were used without further purification. TEMPOL was also bought from Sigma. Sample Preparation and Irradiation. Neat DNA and DNA/ additive (MX/RF) mixtures in different molar ratio were prepared by freeze-drying for homogeneity of the system. Solutions of 20 mg of solid DNA or DNA/additive mixtures in 1 mL of 7 M LiBr (D2O) were prepared which were kept for several days with occasional stirring in darkness and under anaerobic condition. After homogeneous gel-like solutions were obtained, small glass beads were prepared by dropping the

J. Phys. Chem. B, Vol. 110, No. 30, 2006 14977 CHART 1: Structures of Electron Scavenging Additives and TEMPOL As Employed in This Work

solutions with thin head Pasteur pipets into liquid nitrogen. The procedure was kept as close as possible to the one described in ref 29. Pure deoxyribonucleotide samples and mixtures with MX were prepared similarly, only they were not freeze-dried before because of their high solubility in 7 M LiBr (D2O) solution. Samples of the neat additives MX and RF were prepared by dissolving 1 mg of additive in 1 mL of 7 M LiBr (D2O) solution and then forming small beads in liquid nitrogen. Two hundred microliters of 0.87 mM TEMPOL dissolved in 7 M LiBr (D2O) was taken into a 4 mm quartz ESR tube and used as an external standard for spin quantification for all measurements. The samples were generally stored at 77 K. Another solution of TEMPOL (1 mM) was prepared in 7 M LiBr, X-ray irradiated (between 800 Gy and 30 kGy), and stored at 77 K. The temporal dependence of ESR spectra from these samples was subsequently observed. All samples were immersed in liquid nitrogen and irradiated with X-rays (95 kV/25 mA). The pure DNA, pure MX, and DNA/MX samples were X-irradiated to doses of 800 Gy (i.e., 5 min), 1.6 kGy (i.e., 10 min), and 10 kGy (i.e., ∼1 h), respectively. The pure deoxyribonucleotide and the deoxyribonucleotide/MX samples were given X-ray doses of 10 kGy. Pure 7 M LiBr (D2O) was also irradiated (in form of tiny beads) to 800 Gy, 1.6 kGy, and 10 kGy, respectively, for baseline correction. Fricke dosimetry was performed at 800 Gy, and this value was used as basis for estimating higher doses via irradiation time. The irradiated beads of the samples were transferred into 4 mm quartz ESR tubes for the spectral analysis at 77 K. Samples were always stored in the dark and in liquid nitrogen after irradiation. The same conditions were maintained as close as possible for sample preparation, irradiation, measurement, and storage for the purpose of comparative study. ESR Spectroscopy. The first ESR spectra were taken from the samples about 30 min after irradiation. Subsequently, spectra were taken at regular intervals over a time span of about 30 days in total at 77 K. The spectra were recorded on a Bruker ESP 300E instrument using an attenuation of 42 dB (≈12.6 µW microwave power) and 0.16 mT modulation amplitude at

14978 J. Phys. Chem. B, Vol. 110, No. 30, 2006

Pal and Hu¨ttermann

Figure 1. Temporal development at 77 K of first derivative ESR spectra (X-band, ∼9.5 GHz) of LiBr/D2O-glass (7 M) containing 20 mg/mL of a MX:DNA mixture in 1:100 (left panel, spectra A-C) and 1:50 (right panel, spectra D-F) molar ratio of MX with respect to nucleotides, respectively, after X irradiation (10 kGy) at 77 K. Underneath each experimental spectrum the spectrum reconstructed (denoted as rec) from neat DNA and MX patterns along with their goodness of fit is shown (see text). The time of measurement after irradiation is indicated at each experimental trace. The asterisk marks the single line due to MX radicals (cf. Figure 4).

100 kHz frequency. For measurements between 4 and 60 K an ESR 900 cryostat (Oxford Instruments) was used; spectra at 77 K were taken with a quartz “finger” Dewar (Spintec). The ESR spectra of all samples at 77 K were baselinecorrected by subtracting the 7 M LiBr spectrum measured under the same conditions. All measurements were repeated two to five times to verify the reproducibility of the results. ESR spectra of the TEMPOL standard prepared under identical conditions as the samples were always taken before and after measurement of the samples each day. Data Analysis. The spectra were analyzed with the software APOLLO (Dusemund, 1998) for PC providing frequency normalization (in this work 9.5 GHz for X-Band throughout). Apart from procedures such as addition and subtraction of spectra, baseline correction, etc., this program provides for a general least-squares method for the determination of component contributions to composite spectra (denoted “reconstruction” (rec.)) which involves a Gauss-Jordan elimination algorithm.40 The resulting weight of individual components normalized to their respective double integral was used to produce reconstructed spectra. The goodness-of-fit between experimental and reconstructed spectra was calculated according to the formula given by Moens et al.41 Double integral calculations were done with WINEPR (Bruker). The development of component radical concentration with time was analyzed with the program ORIGIN (Originlab Corp., USA) using a nonlinear least-squares fit procedure. Results and Discussion A. Mitoxantrone/DNA. Figure 1 shows ESR spectra of irradiated frozen LiBr glass samples containing DNA together with two different loadings of randomly interspaced MX. The initial spectra (recordings A and D, respectively) were taken about 30 min after irradiation. The subsequent ones (B-F) were recorded after much longer time intervals as indicated at each spectrum. The spectra shown are representative for the temporal development of the whole series of data discussed below. Recordings A-C are for a concentration of one molecule of

MX per 100 DNA nucleotides (denoted 1:100); spectra D-F are for a 1:50 ratio. The spectra were reconstructed from two neat patterns (see below for details), one for DNA and one for MX (the latter marked by an asterisk in the top two spectra of Figure 1). The reconstructed spectra are given underneath each experimental one, and the number attached to the respective recording indicates a very good quality of the reconstruction. From the reconstructions the relative contribution of the MX anion pattern produced is found to be 30% and 45%, respectively, in the initial spectra (A and D) of the systems 1:100 and 1:50 (MX:DNA nucleotides). Thus, MX acts as an electron scavenger under these conditions, as reported, yielding significantly more radicals than expected from the molar ratio.29 The evolution of the spectra components with time shows an increase in relative concentration of the MX anion radical. After about 18 days (spectra C and F, respectively) the fraction of the MX located radicals has increased to about 47% and 62% for the 1:100 and the 1:50 concentration ratios. Thus, the DNA-related contribution has decreased from 70% to about 53% at the ratio of 1:100 and from about 55% to 38% when the ratio of MX was increased to 1:50. This finding is, qualitatively, in line with the previous report although these numbers have not been detailed. The time-dependent variation of the weight of the components associated with MX and DNA in the spectra is dose dependent. The above analysis was performed for three different doses (800 Gy, 1.6 kGy, and 10 kGy, respectively) for both additive concentrations. The component weights at different times are listed in Table 1a showing that there is more change with smaller doses than with 10 kGy.42 For all doses and additive concentrations, however, the trend is similar: the apparent (i.e., relative) contribution of MX radicals to the spectra increases significantly with time. This finding was, we suppose, the basis for suggesting postirradiation electron transfer with time from DNA to MX under these conditions.29 However, the analysis of the total radical concentration of the same set of experimental spectra over the same period of

Response to Ionizing Radiation in DNA

J. Phys. Chem. B, Vol. 110, No. 30, 2006 14979

TABLE 1: Relative Concentration (%) of Radicals at 77 K with Increasing Time in Frozen LiBr Glass Irradiated at 77 K a. Radicals from DNA and from MX in MX:DNA mixtures MX:DNA nucleotide (molar ratio)

dose (kGy)

1:100

0.8 1.66 10

1:50

0.8 1.66 10

time (s)

MX (%)

DNA (%)

goodness of fit

1.8 × 103 1.95 × 105 1.3 × 106 2.9 × 103 1.9 × 105 1.6 × 106 1.8 × 103 3.6 × 105 1.5 × 106 1.8 × 103 1.9 × 105 1.2 × 106 3 × 103 1.9 × 105 1.6 × 106 1.8 × 103 2.5 × 105 1.3 × 106

29 53 61 31 50 62 30 42 47 45 63 69 50 67 73 44 56 62

71 47 38 69 50 38 70 58 53 55 37 31 50 33 27 56 44 38

0 0.035 0.207 0.1111 0.3017 0.0818 0.0 0.4456 0.5183 0 0.0799 0.0926 0 0.0132 0.0992 0.0462 0.1503 0.1972

b. As in (a) but for the RF:DNA system irradiated with 10 kGy RF:DNA nucleotide (molar ratio) 1:100 1:50

time (s)

RF (%)

DNA (%)

goodness of fit

1.8 × 103 5.0 × 105 1.5 × 106 1.8 × 103 4.5 × 105 1.6 × 106

28 47 56 40 60 65

72 53 44 60 40 35

0.0882 0.1769 0.1533 0.0464 0.1832 0.3353

c. As in (a) but for MX: Deoxyribonucleotide (TMP/ dCMP) Mixtures Irradiated with 10 kGy MX:nucleotide

molar ratio

MX:TMP

1:50 1:10

MX:dCMP

1:10

time (s)

MX (%)

nucleotide (%)

goodness of fit

2.4 × 103 2.7 × 10 6 1.8 × 103 2.5 × 106 1.8 × 103 2.7 × 106

4 12 12 26 8 7

96 88 88 74 92 93

0.072 0.45 0 0.238 0 0.078

time displays a considerable decay of radicals at 77 K. The corresponding data are shown in Figure 2 (top). For the two concentrations of MX vs DNA discussed above, the total amount of free radicals is given as the respective double integral value of the experimental spectra. Apart from the dose of 10 kGy, the data for 800 Gy for the two concentrations are included. The results for 1.6 kGy are omitted in order to avoid overcrowding of data points; all three doses will be referred to in the discussion and tables. The normalization for each set of data is to the first spectrum taken after irradiation (about 30 min). As control, the normalized spin concentration (double integral) of the TEMPOL standard is displayed, which is seen to be constant with time, as expected. The decay of the radicals with time at 77 K appears to exhibit approximately two different regimes under all conditions (concentration, dose) which could be termed a “fast” and a “slow” decay regime. The first is responsible for the loss of about 35% of the initial radical population within approximately 2 days. There are slight differences in the data relating to the MX and DNA concentration ratios and the irradiation doses, but the general picture is unaffected by these parameters. Altogether, about 50% to 60% of the initial total radical

Figure 2. (top) Development of total radical concentration with time of MX:DNA mixtures at 77 K given as double integral (DI) values normalized to the first measurement ∼30 min after X-ray irradiation in comparison with an external standard (TEMPOL, closed rhombic points). Data from experiments with two different loadings of MX: DNA (nucleotides) irradiated with two different doses are shown: 1:100/800 Gy (open hexagons); 1:50/800 Gy (crossed hexagons); 1:100/ 10 kGy (filled hexagons); 1:50 /10 kGy (half filled hexagons). (bottom) Delineation of the above data into MX and DNA radical components and their development with time using the respective neat patterns for reconstruction (see text). The convention of symbols used for different additive loadings and doses is same as above but separating the hexagons into circles for MX and squares for the DNA contributions, respectively. The solid lines represent a biexponential fit to the experimental data (see text and tables).

population is lost within the total time period of observation. This finding is a new and essential piece of information for the MX/DNA system. It invokes the need of further exploration of the system since the putative electron transfer from DNA anions to MX must be weighed against the total radical decay. Thus, instead of electron transfer, an alternative explanation for the observed relative shift in fractions of MX and DNA located radicals with time in the experimental spectra could be differential decay behavior of the two constituting radical parts in the aqueous low-temperature LiBr glass matrix. This topic is addressed subsequently. Consider first the decay of free radicals from neat DNA in the LiBr glass under the same conditions. The initial EPR spectra from DNA for two irradiation doses (800 Gy and 10 kGy, respectively) together with their respective patterns after a long storage time at 77 K are shown in Figure 3, top part. The patterns do not change significantly with time or dose, but the signalto-noise ratio decreases with time for both doses. In the bottom part of Figure 3 the decay of the DNA radical concentration is plotted vs time for three different doses. As for the mixed MX/ DNA systems discussed above, one finds roughly a fast and a slow component for the decay. About 70% to 80% of all DNAlocated free radicals are lost within the period of observation. The corresponding results obtained from neat MX in LiBr glasses are shown in Figure 4. As for DNA, the shape of the



Figure 3. (top) Representative ESR spectra of pure DNA (20 mg/1 mL of 7 M LiBr/D2O) irradiated at 77 K with 10 kGy (solid lines) or 800 Gy (dashed lines). The top two recordings are for a short period of time and the bottom two are for a long period of time after irradiation showing that the DNA derived pattern is nearly independent of time and dose. (bottom) Development of the total DNA radical concentration with time at 77 K. Data are shown for the irradiation doses: 800 Gy (open squares); 1.6 kGy (half filled squares); 10 kGy (crossed squares). The solid lines represent biexponential fits to the data. The external concentration standard (TEMPOL) results are shown by rhombic points (filled).

spectra of the MX radical is found to be stable with time and independent of the irradiation dose (top part of Figure 4). Likewise, the concentration of MX radicals also shows decay with time although at a lower rate in comparison to neat DNA system. Between 30% and 40% of all MX radicals are lost over the whole period of observation. However, like in DNA, the decay appears to allow for a division in terms of a fast and a slow time regime. The two sets of data can be analyzed in terms of biexponential decay behavior in order to make a numerical comparison possible using the equation

y ) y0 + A1(exp(-x/t1)) + A2(exp(-x/t2))

(1)

in which x represents the time and y the radical concentration. The respective fitting parameters A and t obtained for the three lines through the experimental data shown above for DNA in Figure 3 are listed in Table 2b. As expected from the experimental behavior, the times t1 and t2 representing the fast and the slow radical decay component, respectively, differ by about a factor of 40-70 supporting the validity of the biexponential analysis. The same analysis for MX decay data gives the fitting results shown for the three doses in the bottom part of Figure 4. The respective time constants and the other parameters are also listed in Table 2b. The two values, t1 and t2, differ again by a factor of 30-40 in magnitude. More importantly, the constants and the weight for the fast and the slow components for DNA and

Figure 4. (top) Same data as in Figure 3 but for pure MX (1 mg/1 mL of 7 M LiBr/D2O). (bottom) Same data and analysis as in Figure 3 but for neat MX.

for MX differ markedly yielding a more effective radical loss with time for DNA. Therefore, if the two components in the mixed DNA/MX system would decay independently (i.e., with the same constants as in the neat systems), an increasing preponderance of MX located radicals with time would be expected. The component decay behavior in the mixed MX/DNA system was analyzed by the spectra reconstruction method. For each of the experimental spectra a numerical reconstruction was performed by using the neat patterns from the two constituting components. This gives the fraction of MX and of DNA located radicals contributing to the respective spectrum. The analysis is facilitated and justified by the fairly high stability of the component patterns with time illustrated in Figures 3 and 4, respectively, and from the fact that the component spectra can be separated easily due to their strongly different appearance. The result of the reconstructions was shown already for selected spectra in Figure 1 proving the feasibility of the method. The breakdown of the total data set shown in Figure 2 (top part) into component contributions for the two concentrations of MX with respect to DNA in the MX/DNA mixture as well as for the two doses is displayed in the bottom part of Figure 2. The lines show the curves fitted to eq 1 for the two components under each experimental condition. The numerical values for the decay time constants gained from the analysis are listed in Table 2a. Without discussion of the numbers, it is clear already from the visual comparison of the bottom parts of Figures 3 and 4 with that of Figure 2 that the decay behavior of the components in the mixed system strongly resembles that of each respective component in its neat state. Although the reconstruction is prone to some small error, the alignment of decay constant values between the individual and the combined system is so close that the conclusion is warranted that the decay of each component in the mixed system is nearly fully independent from the presence of the respective second constituent. The



TABLE 2: Parameters A1, t1, A2, and t2 and the MX Radical Fractions

a. Parameters A1, t1, A2, t2 Obtained from Fitting the Experimental Time-Dependent Decay Data at 77 K to y ) y0 + A1(exp(-x/t1)) + A2(exp(-x/t2)) for the DNA and the MX Radical Fractions Obtained from Spectra Reconstructions in the Frozen Glass System of DNA:MXa,b MX:DNA nucleotide dose radical A1 t1 (s) (molar ratio) (kGy) fractions (% err) (% err) 1:100

0.8

DNA

0.552 (4%) 0.117 (32%) 0.514 (11%) 0.152 (7.2%) 0.465 (4%) 0.171 (9%)

MX 1.66

DNA MX

10

DNA MX

11560 (10%) 5622 (28%) 6392 (24%) 9303 (30%) 11802 (8%) 17953 (17%)

A2 (% err)

t2 (s) (% err)

0.352 (5.4%) 0.216 (5%) 0.370 (8%) 0.089 (12.3%) 0.301 (4%) 0.078 (9%)

422606 (11%) 1833463 (15.7%) 197196 (13.8%) 1012195 (34%) 981732 (10%) 808241 (24%)

MX:DNA nucleotide dose radical A1 A2 t1 (s) (molar ratio) (kGy) fractions (% err) (% err) (% err) 1:50

0.8

DNA

0.519 (5%) 0.111 (16%) 0.595 (13%) 0.144 (21%) 0.360 (10%) 0.193 (8%)

MX 1.66

DNA MX

10

DNA MX

14305 (12%) 16742 (36%) 5112 (23%) 9798 (35%) 8376 (26%) 10491 (26%)

0.319 (6%) 0.152 (8%) 0.372 (6%) 0.187 (7.5%) 0.318 (10%) 0.175 (7.4%)

t2 (s) (% err) 706909 (12%) 1690474 (18%) 207066 (13.4%) 663608 (18%) 257182 (18.2%) 1175019 (18%)

b. As in (a) but for Direct Experimental Data from Pure DNA, Pure MX, and Pure RFc system

dose (kGy)

A1 (% err)

t1 (s (% err)

A2 (% err)

t2 (s) (% err)

pure DNA

0.8

0.787 (6%) 0.787 (6.5%) 0.543 (5%)

4522 (13%) 4470 (15%) 9492 (12%)

0.284 (7.4%) 0.212 (11.3%) 0.241 (9%)

296581 (15%) 203999 (23%) 671485 (18.6%)

1.66 10

system

dose (kGy)

A1 (% err)

t1 (s (% err)

A2 (% err)

t2 (s) (% err)

pure MX

0.8

0.192 (18%) 0.291 (6.2%) 0.177 (9%) 0.260 (20%)

8272 (34%) 26306 (17%) 13567 (22%) 4107 (25.2%)

0.2 (12.5%) 0.152 (9.2%) 0.231 (6.5%) 0.195 (9.7%)

527794 (17%) 699965 (21.6%) 416383 (12%) 219310 (24.5%)

1.66 10 pure RF

10

c. As in (a) but for Reconstructed Radical Fractions from the RF:DNA Systems (dose 10 kGy)d RF:DNA nucleotide (molar ratio)

fractions

1:100

DNA RF

A1 (% err)

t1 (s) (% err)

A2 (% err)

t2 (s) (% err)

0.524 (6%) 0.187 (9%)

6631 (15%) 9774 (21%)

0.285 (8%) 0.160 (7.5%)

301781 (16.4%) 766002 (20%)

RF:DNA nucleotide (molar ratio) 1:50

fractions DNA RF

A1 (% err)

t1 (s) (% err)

A2 (% err)

t2 (s) (% err)

0.579 (8%) 0.220 (14%)

4116 (19%) 9955 (36%)

0.311 (9%) 0.141 (15%)

104427 (16.2%) 657725 (34%)

d. As in (a) but for Deoxyribonucleotides TMP and dCMP as for their Mixtures with MX (dose 10 kGy)e system

molar A1 t1 A2 t2 ratio fractions (% err) (s) (% err) (% err) (s) (% err)

pure TMP MX:TMP

TMP 1:10

TMP MX

0.632 (2.4%) 0.418 (11%) 0.077 (31%)

6731 (6.2%) 9742 (25%) 6192 (28%)

0.194 (6%) 0.408 (7.5%) 0.2425 (2%)

446932 (10%) 750571 (16%) 949923 (5.2%)

system

molar ratio fractions

pure dCMP

dCMP

MX:dCMP 1:10

dCMP MX

A1 (% err) 0.271 (10.33%) 0.285 (11.2%) 0.278 (13.3%)

t1 A2 t2 (s) (% err) (% err) (s) (% err) 12880 (20%) 8104 (21%) 7053 (27%)

0.273 (6.2%) 0.287 (7%) 0.284 (10%)

962421 (13.2%) 568649 (12.35) 322705 (16.4%)

a The relative error (in %) for each parameter is shown in parentheses as calculated from absolute errors obtained from nonlinear least-squares fitting routines used by ORIGIN. b The R2 value showing the goodness of fit varies in range of 0.98998 ( 0.01129. c R2 values ranging 0.992 ( 0.00734. d R2 values ranging 0.992 ( 0.00853. e R2 values ranging 0.99347 ( 0.00344.

observed shift in fractions of components with time (cf. Figure 1) can thus be ascribed to result dominantly from differential radical stability for DNA and MX located radicals, respectively. In the postirradiation period there is no discernible communication within the system DNA and scavenger-like electron transfer involved. B. Riboflavin/DNA. To enhance the understanding of the postirradiation behavior of DNA radicals in low-temperature glasses, we chose the system of DNA mixed with different loadings of riboflavin (RF). This compound is known to be no intercalator but to form a close association with DNA.39 As a consequence one would not expect electron transfer along the DNA strand (intrastrand transfer) from the beginning, and independent component decay within a mixture should perhaps prevail. The experimental spectra for one of the radiation doses used before (10 kGy) are shown in Figure 5 for the concentration ratio of one RF molecule per 100 nucleotides (1:100, left

column) and for the ratio 1:50 (right column), as employed before in the MX/DNA system. As before, the reconstructions shown under the experimental spectra are made for a twocomponent system, the pattern of DNA being the same as used in the MX/DNA system. The RF pattern will be shown below. Unlike the situation with MX, there seems to be only a small influence of the additive RF in the initial spectra (A and D, respectively, in Figure 5). Cursory visual inspection might lead to the conclusion that the basic doublet type pattern expected for DNA is by-and-large retained. Quantitative reconstruction, however, shows that about 28% of the radicals are located on RF in spectrum A and about 40% in spectrum D. These numbers are very close to those found above for the MX located fraction at the same concentration ratios. Also, the change of the spectra with time at 77 K gives a similar increase in additive located radical fractions. At the end of the observation series, about 60% of the radicals are on the additive RF for the concentration



Figure 5. Temporal development of first derivative ESR spectra (X-band, ∼9.5 GHz) of LiBr/D2O-glass (7 M) containing 20 mg/mL of RF: DNA (nucleotides) in 1:100 (left panel, spectra A-C) and 1:50 (right panel, spectra D-F) molar ratio of RF to nucleotides, respectively, after X irradiation (10 kGy) at 77 K. Underneath each experimental spectrum the spectra reconstructed (denoted as rec) from neat DNA and RF patterns along with their goodness of fit are shown (see text). The time of measurement after irradiation is indicated at each experimental trace.

ratio 1:100 (spectrum C). The respective value for the 1:50 concentration is 65% (spectrum F). Both values are again in close proximity to those found in the MX/DNA systems as can be inferred from parts a and b of Table 1. Altogether, the nonintercalating compound RF participates like an apparent electron scavenger in the distribution of free radicals formed during irradiation. The reason for the apparent lack of strong changes in the experimental spectra when compared to Figure 1 derives from the differences between the neat MX radical pattern which is a “sharp” singlet (i.e., with a small line width) and the spectrum for riboflavin electron adduct radicals. The latter is shown in Figure 6 as obtained directly (30 min) after irradiation and after several weeks of storage. It is seen to be a broad singlet with some shoulders. Due to the width of this signal, small amplitude changes will result in large double integral variations. The bottom part of Figure 6 shows the decay of RF derived radicals in the LiBr low-temperature glass with time when stored at 77 K. Again one can fit the data points to a biexponential curve yielding a fast and a slow component for the decay of the RF radicals. The results of the numerical analysis are also listed in parts b and c of Table 2. The change of the total radical concentration in the system containing both DNA and RF is shown in Figure 7, top part. As discussed for the MX/DNA case, considerable fading with time at 77 K is observed which displays a fast and a slow time component, the former leading to loss of about 35% of the radical population within the first day. This value is fairly independent of the RF concentration ratio which is 1:100 nucleotides and 1:50, respectively. Following the procedure described above for MX, the individual component fading was analyzed from the reconstructions of the experimental spectra. The result is given in Figure 7 (bottom part) for the two concentration ratios (1:50 and 1:100, respectively). As with MX, the fading of DNA radicals in the RF/DNA mixture is faster and more efficient than that of the RF radicals so that differential radical stability is the origin of the enhanced fraction of RF

Figure 6. (top) ESR spectra of pure RF (1 mg per 1 mL of 7 M LiBr/ D2O) X-ray irradiated (10 kGy) and measured at 77 K. The qualitative feature of the spectra shows no variance with dose and time. (bottom) Development of concentration of pure RF radicals in frozen LiBr glass at 77 K (10 kGy dose) with time. The biexponential fit to the experimental points is shown as a solid line. The external standard data (TEMPOL) as a control are shown by rhombic points (filled).

radicals with time. Indeed, the time constants for DNA derived from the biexponential decay fitting (listed in Table 2) are approximately the same as those found in the MX/DNA mixture (and in neat DNA) so that, again, the dominant process governing the spectral changes of the mixed system in the postirradiation period is differential decay.


Figure 7. (top) Development of total radical concentration with time of RF: DNA mixtures at 77 K given as double integral (DI) values normalized to the first measurement after X-ray irradiation (∼30 min) in comparison with an external standard (TEMPOL, closed rhombic points). Data from experiments with two different loadings of RF: DNA (nucleotides) irradiated each with 10 kGy are shown: 1:100 (closed hexagons); 1:50 (half filled hexagons). (bottom) Delineation of the above data into RF and DNA radical components and their development with time using the respective neat patterns for reconstruction (see text). The convention of symbols used for different additive loadings and doses are same as above but separating the hexagons into pentagons for RF and squares for the DNA contributions, respectively. The solid lines represent a biexponential fit to the experimental data (see text and tables).

C. Deoxyribonucleotides/MX. As a case for “a priori” expected independence of component radical decay, the combinations of MX with the deoxyribonucleotides TMP and dCMP were analyzed. In these systems there should, in principle, be no association between either component. Rather, the LiBr glass is expected to provide for a homogeneous distribution of both the deoxyribonucleotide and the MX molecules in the matrix. The two pyrimidine deoxyribonucleotides were chosen since their anions are the ones expected to be formed in DNA in the low-temperature LiBr glass.29 The experimental spectra obtained under these conditions are given in Figure 8. For TMP (left column), three groups of spectra are shown. One is for the neat compound the others are for the concentration ratios 1:50 and 1:10 molecules MX vs TMP. For each group, the top recording is for the initial spectrum taken about 30 min after irradiation whereas the second spectrum reflects the development of experimental patterns with time and is taken after storage of the samples for about 1 month. The right column gives the corresponding spectra for dCMP and its mixtures with MX. For both deoxyribonucleotides it is noted that the initial spectra have hardly relevant MX radical contributions. Indeed, the corresponding numbers listed in Table 1c show that the contributions of MX radicals to the total radical concentration are very close to its stoichiometric value. In the 1:10 mixture for TMP the MX radical fraction amounts to 12%, in the 1:50 mixture to about 4.3%. A similar result is obtained for the mixture with

J. Phys. Chem. B, Vol. 110, No. 30, 2006 14983 dCMP. These results imply that there is no influence of MX on the free radical distribution in these systems during irradiation, a situation completely different to that observed in MX/ DNA mixtures above. This result therefore supports, in retrospect, the assumption implicitly made above that there is a very close association between the DNA and the additives MX and RF. The decay of the total radical concentration in the same samples of TMP with time is shown in Figure 9. Again one finds loss of radicals at 77 K in terms of a fast and a slow component. The difference between neat TMP and the mixtures with MX in the ratio 1:10 and 1:50, respectively, are seen to be small. In total, about 70% of the initially detected radicals are lost at the end of the time series. The reconstruction of the mixtures between TMP and MX in terms of individual component decay gives the results displayed in the bottom part of Figure 9. Again, the rate of decay for the TMP located radicals is significantly faster than that of the MX fraction leading to an enhanced visibility of the latter group in the spectra with time as observed in the experiment (cf. Figure 8, left column). The results obtained from dCMP are different. The experimental spectra of Figure 8 (right column) showed no significant difference between the initial spectra and the recordings at the end of the observation time in terms of fractions of MX contributing vs dCMP radicals. The decay of the total radical concentration is also quantitatively different. As shown in Figure 10 (top part) the loss of radicals involves only about 20% on the first day and settles at about 50% at the end of the observation time. These numbers are fairly independent of the amount of MX present in the mixture. The reconstruction analysis shown in the bottom part gives about the same decay behavior for dCMP and MX for the 1:10 concentration. It must be concluded that the temporal stabilities of MX radicals and species located on dCMP at 77 K are about equal. Again, there is no indication of any transfer from the nucleotide to MX. D. Mechanistic Aspects; Postirradiation Radical Decay. For both MX and RF when associated with DNA, their fraction of radicals found immediately after irradiation at 77 K is by far (a factor of about 20-30) larger than expected from their stoichiometric ratio. The amount of enhancement is comparable for both additives. Since they associate differently with DNA, the specific mode of binding appears to be irrelevant for this effect. Probably, an unspecific “close” association is required. Similar results are found in solid, lyophilized DNA containing the respective additive or in frozen aqueous solutions. This type of “intrairradiation” transfer between DNA bases and closely associated additives has been known for a long time in the field, and there are numerous examples for it. One of the first descriptions was presented in 1970.43 A recent report deals with the mixture of DNA with histones in chromatin in which DNA takes up about a factor of 2 more radicals than expected from the mass ratio.38 Other recent examples concern the efficient and selective hole trapping by minute amounts of thiosubstituted nucleic acid bases doped into single crystals of the corresponding unsubstituted base44 or by doping 5-methylcytosine into the cytosine crystalline lattice.45 Despite many examples, the details of the mechanism(s) governing the radical distribution between DNA located radicals and additive radicals in the intrairradiation phase are not fully clear. The main impact from this report concerns, on the other hand, the absence of significant net electron transfer with time at 77 K from DNA radicals to the additive(s) in the postirradiation period. This finding not only is in direct conflict with earlier



Figure 8. (left panel) The initial (30 min) and final (∼4 weeks) first derivative ESR spectra of a time series of spectra obtained after X-ray irradiation (10 kGy) from pure TMP (top), a mixture of MX:TMP of 1:50 (middle), and 1:10 (bottom), respectively. (right panel) The respective experimental spectra obtained from dCMP and its mixtures with MX.

interpretations of the same system29,31 but also appears unexpected since electron-transfer, e.g., in proteins is very well established.46,47 Moreover, the substantial amount of radical decay, especially of the DNA-located electron adducts in the glass, clearly shows that postirradiation processes occur with time, e.g., at 77 K, which modulate the observed radical balance. To our knowledge, no mechanism has been put forward so far which can be applied to explain the observed radical decay. Oxidation of the electron adducts on DNA and on the additives by Br2- might be considered: This would imply that the trapped holes should be “somewhat” mobile at 77 K and even at lower temperatures. Typically, the Br2- species become mobile and react from about 140 K onward (see below), but they might perhaps be detrapped partially at lower temperatures. However, two experimental observations and one general argument discussed below appear to rule out this type of mechanism. As an alternative scheme we consider that, apart from the electrons which add to the DNA (pyrimidine) bases and to the additives and apart from the holes trapped as Br2- in the LiBr glass, another fraction of electrons and holes exists. These are denoted “defects” and they are supposed to remain trapped in the matrix (e.g., at 77 K) when the irradiation has ended. It is this newly proposed fraction, external to the DNA/additive or solute system, which could govern the postirradiation reactions. Three key properties are then needed for these matrix-trapped defects. First, they must be ESR-mute since they are simply not detected under the conditions applied. Second, their energy should be such that they are mostly inept to form new radicals but rather capable of destroying them. Third, their number should be by far larger than that of the free radicals stabilized at 77 K. The simple picture then evolves that storage of the irradiated samples at 77 K leads to liberation of the defects from their (shallow) matrix-located traps with time. Apart from recombinations which go unnoticed by ESR, their reactions then lead to the observed decay of the ESR-active free radical population. This process then should be dominating the postirradiation time profile of radicals. The one proposed earlier, electron transfer from a DNA

Figure 9. (top) Development of total radical concentration with time of TMP and MX:TMP mixtures at 77 K given as double integral (DI) values normalized to the first measurement (∼30 min) after X irradiation (10 kGy) in comparison with an external standard (TEMPOL, closed rhombic points). Pure TMP (open hexagons); MX:TMP (1:50) (half filled hexagons); MX:TMP (1:10) (crossed hexagons). (bottom) Delineation of the above data for MX:TMP (1:10) (crossed hexagons in top part) into TMP (crossed triangles) and MX (crossed circles) radical components and their development with time using the respective neat patterns for reconstruction. The solid lines represent biexponential fits to the experimental data (see text and tables). The data for pure TMP are included for comparison (open hexagons)


Figure 10. (top) Same plot as in Figure 9 (top) but for dCMP and its mixtures with MX. Nomenclature of symbols is transferred from Figure 9. (bottom) Same delineation of MX and nucleotide radical contributions for MX:dCMP (1:10) as in Figure 9 but for dCMP as nucleotide. The data for pure dCMP are included for comparison (open hexagons).

pyrimidine radical anion to MX or another additive via some kind of transport structure,29 may perhaps occur but has, if at all, a negligible quantitative effect on the ratio of free radical types detected.48,49 With this model the well-established general picture of electron-transfer remains unconcerned. Only, its contribution is insignificant in an environment loaded with trapped-radiation-produced defects. Consider first the behavior of Br2- in the postirradiation phase. It can be tested by using TEMPOL as an ESR-active monitor. Figure 11 (part A) gives a spectrum (a) for the (stable) TEMPOL radical (1 mM) measured at 77 K. When irradiated to a dose of 30 kGy, its ESR activity is apparently fully abolished (spectrum b) by reaction with radiation-produced electrons whereas the holes form Br2- as usual. After about 30 days, the typical time range used in the above radical decay studies, a very minute TEMPOL activity was gained back (not shown). After 65 days of storage at 77 K some additional ESR activity of TEMPOL was found to be restored (spectrum c). From a dilution series of TEMPOL a concentration of roughly 5-8 µM can be assigned to this latter spectrum (reference spectrum d), a number which must be kept with reservation due to the large errors involved in double integration of the weak signals. Nevertheless, Br2- apparently cannot reactivate the TEMPOL radical within up to about 65 days at 77 K to a significant extent. That this is not due to a lack of oxidative power which can be derived from the data given in Figure 11B. Irradiation of TEMPOL with doses lower than 30 kGy yields only partial loss of the ESR activity; e.g., a 600 Gy spectrum corresponding to about 60% of the control activity is obtained. Storage of this sample at 77 K leaves the ESR activity unchanged over a period of 30 days (data not shown). Thus, both the active TEMPOL fraction and its ESR-mute product produced from irradiation are highly stable against any reaction


Figure 11. (A) X-band (∼9.5 GHz) ESR spectra of (a) 1 mM TEMPOL frozen in 7 M LiBr/ D2O, (b) same sample X-irradiated with 30 kGy and measured after 30 min, (c) sample as in (b) measured after 65 days, (d) 9.5 µM frozen TEMPOL in 7 M LiBr/D2O as reference. X irradiation, ESR measurement, and storage were always done at 77 K. (B) Loss of ESR activity of TEMPOL (1 mM in 7 M LiBr/D2O) after X irradiation (600 Gy) and measurement at 77 K in LiBr glass with respect to control; effect of thermal annealing of the irradiated sample to the temperatures indicated and subsequent measurement at 77 K on the ESR activity (double integral value) of TEMPOL showing reactions of Br2-.

in the postirradiation time regime at that temperature. However, when annealing to higher temperatures, reactions do occur in the sample. First, between 125 and 160 K a sizable decay of TEMPOL activity (up to about 20% loss) is observed. More important, further annealing enhances the ESR activity. Both processes can be ascribed to reactions of Br2-. The decay could involve oxidation of the radicals, whereas the formation of TEMPOL radicals should be expected to involve oxidation of the diamagnetic product formed upon irradiation. In any case, Br2- is seen to react when it becomes mobile and therefore it should not be connected to a significant extent with the decay observed for DNA and additive radicals at 77 K. One further argument leads to the same conclusion. If Br2were to react with the solute radicals at 77 K, one has to expect, apart from their decay, new free radicals from oxidation of solute molecules which were not yet converted to free radicals by irradiation. Thus, other patterns than those of the electron adduct radicals discussed above should be observed. There is little indication of this process since mostly the patterns remain stable throughout the decay period. Only TMP (Figure 8) is seen to display a change in ESR pattern; some smaller change is also seen in DNA at the end of the storage time. The effect in both cases is so small that it cannot be analyzed quantitatively. The dominant process is just pattern decay. In the same context one can argue that the differential decay of the different free radicals should rule out an involvement of Br2- Apparently, DNA radicals decay more efficiently than those on mitoxantrone or


Figure 12. (top panel): Temporal development of ESR-spectra intensity from frozen glass samples of pure TMP (20 mg/1 mL in 7 M LiBr (D2O)), X-ray irradiated (10 kGy) at 77 K and measured at 10 K (filled squares), 40 K (filled circles), and 77 K (filled triangles), respectively. The open triangles are for TMP in frozen aqueous solutions (50 mg/1 mL in D2O), X-ray irradiated (20 kGy) at 77 K and measured at the same temperature after irradiation. (bottom panel): Temporal development of ESR-spectra intensity from frozen glass samples of neat DNA (20 mg/1 mL in 7 M LiBr (D2O)), X-ray irradiated (10 kGy) at 77 K, and measured at 14 K (filled squares) and at 77 K (filled triangles), respectively. The open triangles are for DNA in frozen aqueous solutions (50 mg/1 mL in D2O) measured at 77 K after irradiation at that temperature.

TEMPOL. If Br2- were involved, one would expect indifferent oxidation perhaps modulated by differential accessibility but not by radical stability. Thus, potential alternative mechanisms for the free radical decay at 77 K and lower temperatures (see below) in the glass matrix must be discussed. Radical-radical recombination between solute radicals must be discarded under these conditions. Therefore, not considered before in the literature, it appears likely that there must be species in the glass which can destroy free radicals with time at 77 K and which are indeed ESR-mute as discussed above. A prominent example for an ESR-mute species is the solvated electron in ice at neutral pH and at 77 K. Only at alkaline pH a sufficiently deep trap structure appears to have evolved allowing for the species to become ESR visible.50,51 A similar situation might apply to the matrix defects discussed here. One aspect of the “defect” mechanism which can be tested experimentally concerns the influence of the temperature on the postirradiation time profile of free radicals. Unlike electron tunneling processes, radical decay should be temperature dependent if the picture of detrapping matrix-trapped defects were reasonable. Only preliminary results are given here for the nucleotide TMP and for DNA. After irradiation at 77 K, the samples were kept at 77 K and at lower temperatures. Figure 12 gives the results. For TMP (top panel) the samples were kept at 10 and at 40 K whereas the DNA (bottom panel) was

Pal and Hu¨ttermann probed at 14 K in addition to 77 K; the temperatures below 77 K were chosen in order to gain high-temperature stability under the given experimental conditions since otherwise the results would show a large scatter. Also, the total time interval applied in this case was only about 10 h so that only part of the “fast component” of decay after irradiation was probed; measurements for longer time periods under these conditions, i.e., with the sample being within the helium cryostat in the ESR apparatus, are not feasible. Nevertheless, the results clearly show that the decay efficiency is temperature dependent for both TMP and DNA; storage temperatures below 77 K reduce the loss of radical activity with time. Interestingly the effect of the temperature reduction on the decay efficiency is larger in TMP than in DNA, although most of the DNA radicals probably are located on TMP sites if judged from the difference in the decay efficiency of the neat nucleotide TMP as compared to dCMP (cf. Figures 9 and 10). This could imply that the accessibility of the free radicals to the defects when liberated from their matrix traps is larger in the isolated deoxyribonucleotide than it is in DNA. The accessibilty factor in both systems, TMP and DNA, can be tested, although probably not fully independent from the other parameters such as trap-depth, by changing the matrix. We have probed the system of frozen aqueous solutions which have been studied quite intensely in the past.36 Roughly, it was proposed that the solute in this case should be confined in “puddles” formed by exclusion from water during its freezing and that these regions were surrounded by bulk-phase ice. As a consequence, the distribution of the solute over the sample volume is considered to be highly inhomogeneous, in contrast to the situation in the glass. Therefore, we expect that the accessibility of the solute free radicals for defects in this situation should be much smaller and thus defect recombination in the bulk ice phases should be the dominant postirradiation process. This is indeed observed experimentally for TMP and DNA as is also shown in Figure 12. The radical decay for both compounds during the fast decay period (first day) in the glass is much less efficient in the frozen aqueous solution at the same temperature (i.e., 77 K). It can, of course, not be excluded, that this effect is also related to a difference in the trap depth of the two systems. Although there is little work available in the literature which can be compared with the present results, some reports seem to be related. For example, the apparent biexponential decay behavior of the fading profiles in all samples tested is not without precedent. A case which could be of close relation with the present findings involves a small trapped ESR active species, the hydrogen atom. Very early on in the field of radiation chemistry the decay kinetics of hydrogen atoms trapped in irradiated sulfuric acid glasses were studied in detail. At 77 K a clear separation between a rapid and a slow decay phase of this species was observed in the postirradiation period which involved time ranges comparable to those used here. It was analyzed in terms of two traps of different depth rather than in terms of nonrandom distance distribution between the H-atom and its reaction partner.52 It could be that, under our conditions, both the apparent fast and the slow components might comprise some distribution of trap energies. Nevertheless, the partition into two phases appears to be operationally a sensible approach. Another early work with potential relevance to the present findings is a study of the decay of radiation produced organic free radicals in solid samples by heat. The time period of measurement applied in that work was only of the order of 1 h or less. Under these conditions, monoexponential decay was

Response to Ionizing Radiation in DNA found for solid samples of organic acids or amino acids which were irradiated at 300 K and subsequently heated from 100 to 150 °C. It was proposed that mobile charges were induced by heat in the organic solid, unnoticeable by ESR perhaps due their low concentration. These charges were suggested to interact with the organic free radicals in terms of destruction by site hopping.53 The picture of radiation produced defects in the solid responsible for free radical decay was not elaborated but can, in our view, also explain the above results. Conclusion In the system of DNA associated with additives such as MX and RF in low-temperature glasses, the response to ionizing radiation can be divided into two phases, the intra- and the postirradiation period. In the former, there is a strong influence of the additives on the distribution of radicals between the DNA and the additive component observed by ESR spectroscopy in favor of the additive. The postirradiation phase, on the other hand, is governed by processes which induce net decay of the free radical population with time. Differential stability of the radicals against the decay then changes the relative contribution of DNA vs additive radicals in the postirradiation period. Electron tunneling from DNA to the additive, unlike earlier suggestions in the literature, is not a process involved in this change to any significant amount. A detailed mechanistic description of the decay process is still not possible. A proposal has been given here which involves detrapping of matrixstabilized ESR-mute defects formed upon ionizing irradiation. Some of the potential parameters connected with such a mechanism have been tested, and the results have shown the feasibilty of this proposal but further studies are needed for detailed quantitative assessments. Acknowledgment. The help of J. Marx with the X-ray irradiation facility is gratefully acknowledged. Thanks are also due to Dr. R. Kappl and to H. Luxenburger for providing help with useful discussions and suggestions. References and Notes (1) Watson, J.; Crick, F. Nature 1953, 171, 737. (2) Eley, D. D.; Spivey, D. I. Trans. Faraday Soc. 1962, 58, 411. (3) For a survey at an early stage see, e.g.: Effects of Ionising Radiation on DNA; Bertinchamps, A. J., Hu¨ttermann, J., Ko¨hnlein, W., Teoule, R. Eds.; Springer: Berlin, 1978. (4) Bernhard, W. A. AdV. Radiat. Biol. 1981, 9, 199. (5) Hu¨ttermann, J. Ultramicroscopy 1982, 10, 25. (6) Symons, M. C. R. J. Chem. Soc., Faraday Trans. 1 1987, 83, 1. (7) Visscher, K. J.; Hom, M.; Loman, H.; Spoelder, H. J. W.; Verberne, J. B. Radiat. Phys. Chem. 1988, 32, 465. (8) Candeias, L. P.; O’Neill, P.; Jones, G. D. D.; Steenken, S. Int. J. Radiat. Biol. 1992, 61, 15. (9) Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.; Bossmann, S. H.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025. (10) Kelly, S. O.; Barton, J. K. Science 1999, 283, 375. (11) Giese, B.; Amandrut, J.; Ko¨hler, A.-K.; Spormann, M.; Wessely, S. Nature 2001, 412, 318. (12) Harriman, A. Angew. Chem., Int. Ed. Engl. 1999, 38, 945. (13) Porath, D.; Bezryadin, A.; de Vries, S.; Dekker, C. Nature 2000, 403, 635.

J. Phys. Chem. B, Vol. 110, No. 30, 2006 14987 (14) Debije, M. G.; Milano, M. T.; Bernhard, W. A. Angew. Chem., Int. Ed. 1999, 38, 2752. (15) Priyadarshy, S.; Risser, S. M.; Beratan, D. N. J. Phys. Chem. 1996, 100, 17678. (16) Turro, N. J.; Barton, J. K. J. Biol. Inorg. Chem. 1998, 3, 201. (17) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1982, 811, 265. (18) Ratner, M. Nature 1999, 397, 480. (19) Jortner, J.; Bixon, M.; Langenbacher, T.; Michel-Beyerle, M. E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12759. (20) Handerson, P. T.; Jones, D.; Hampikian, G.; Kan, Y.; Schuster, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8353. (21) Olson, E. J. C.; Hu, D.; Hormann, A.; Barbara, P. F. J. Phys. Chem. 1997, 101, 299. (22) Anderson, R. F.; Wright, G. A. Phys. Chem. Chem. Phys. 1999, 1, 4827. (23) Yu, Z. G.; Xueyu S. Phys. ReV. Lett. 2001, 86, 6018. (24) Ly, D.; Sanii, L.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121, 9400. (25) Nunez, M. E.; Noyes, K. T.; Barton, J. K. Chem. Biol. 2002, 9, 403. (26) Weiland, B.; Hu¨ttermann, J. Int. J. Radiat. Biol. 1998, 74, 341. (27) Pezeshk, A.; Symons, M. C. R.; McClymont, J. D. J. Phys. Chem. 1996, 100, 18562. (28) Hu¨ttermann, J. In Radical Ionic Systems; Lund, A., Shiotani, M., Eds.; Kluwer Academic: Dordrecht, 1991; p 435. (29) Messer, A.; Carpenter, K.; Forzley, K.; Buchanan, J.; Yang, S.; Razskazovskii, Y.; Cai, Z.; Sevilla, M. D. J. Phys. Chem. B 2000, 104, 1128. (30) Cai, Z.; Gu, Z.; Sevilla, M. D. J. Phys. Chem. B 2000, 104, 10406. (31) Cai, Z.; Li, X.; Sevilla, M. D. J. Phys. Chem. B 2002, 106, 2755. (32) Cai, Z.; Sevilla, M. D. J. Phys. Chem. B 2000, 104, 6942. (33) Cai, Z.; Li, X.; Sevilla, M. D. J. Phys. Chem. B 2002, 106, 2755. (34) Cai, Z.; Gu, Z.; Sevilla, M. D. J. Phys. Chem. B 2001, 105, 6031. (35) Cai, Z.; Sevilla, M. D. In Topics in Current Chemistry; Schuster, G. B., Ed.; Spinger Verlag: Berlin, 2004; Vol. 237, p 103. (36) Sevilla, M. D.; Becker, D. In Electron Paramagnetic Resonance, Specialist Periodical Reports; Gilbert, B. C., Davies, M. J., Murphy, D. M., Eds.; The Royal Society of Chemistry: Cambridge, 2004; Vol. 19, p 243. (37) Weiland, B.; Hu¨ttermann, van Tol, J. Acta Chem. Scand. 1997, 51, 585. (38) Weiland, B.; Hu¨ttermann, J. Int. J. Radiat. Biol. 2000, 76, 1075. (39) Ito, K.; Inoue, S.; Yamamoto, K.; Kawanishi, S. J. Biol. Chem. 1993, 268, 13221. (40) Press, W. H.; Teukolsky, S. A.; Vetterling, W. T.; Flannery, B. P. Numerical Recepies in C: The Art of Scientific Computing, 2nd ed.; Cambridge University Press: Cambridge, 1992; p 671. (41) Moens, P.; de Volder, P.; Hoogewus, R.; Callens, F.; Verbeck, R. J. Magn. Reson., Ser. A 1993, 101, 1. (42) The dose value of 800 Gy corresponds most closely with that used in ref 30. (43) Gregoli, S.; Taverna, C.; Bertinchamps, A. Int. J. Radiat. Biol. 1970, 18, 577. (44) Herak, J. N.; Sankovic, K.; Hole, E. O.; Sagstuen, E. Phys. Chem. Chem. Phys. 2000, 2, 4971. (45) Krivokapic´ A.; Sagstuen, E J. Phys. Chem. A 2003, 107, 9561. (46) Gray, H. B.; Winkler, J. R. Annu. ReV. Biochem. 1996, 65, 537. (47) Gray, H. B.; Winkler, J. R. Proc. Natl. Acad. Sci. 2005, 102, 3534. (48) Note that recent work by Barton and co-workers has proposed that charge transfer in a glassy matrix at 77 K does not occur in DNA due to lack of motional flexibility of the polymer (see ref 49), but this thesis cannot be tested under the present conditions. (49) O’Neill, M. A.; Barton, J. K. J. Am. Chem. Soc. 2004, 126, 13234. (50) Eiben, K. Angew. Chem., Int. Ed. Engl. 1970, 9, 619. (51) Schulte-Frohlinde, D.; Eiben, K. Z. Naturforsch. A: Astrophys., Phys. Phys. Chem. 1962, 17, 445. (52) Sprague, E. D.; Schulte-Frohlinde, D. J. Phys. Chem. 1973, 77, 1222. (53) Horan, P. K.; Taylor, W. D.; Strother, G. K.; Snipes, W. Biophys. J. 1968, 8, 164.

Postirradiation Electron Transfer vs Differential Radical Decay in X

Recommend Documents