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J. Phys. Chem. B 1997, 101, 1460-1467
Competitive Electron Scavenging by Chemically Modified Pyrimidine Bases in Bromine-Doped DNA: Relative Efficiencies and Relevance to Intrastrand Electron Migration Distances Yurii Razskazovskii,† Steven G. Swarts,‡ Joseph M. Falcone,‡ Craig Taylor,† and Michael D. Sevilla*,† Department of Chemistry, Oakland UniVersity, Rochester, Michigan 48309, and Department of Radiation Oncology, Bowman Gray School of Medicine of Wake Forest UniVersity, Medical Center BouleVard, Winston-Salem, North Carolina 27157 ReceiVed: October 8, 1996; In Final Form: December 2, 1996X
ESR spectroscopy at low temperatures is employed to investigate electron transfer within DNA doped with randomly spaced electron traps. The traps were introduced by careful bromination of DNA in ice-cooled aqueous solution. The procedure is shown by NMR and GC/MS techniques to modify thymine, cytosine, and guanine 2′-deoxyribosides, transforming them into 5-bromo-6-hydroxy-5,6-dihydrothymine, T(OH)Br, 5-bromocytosine, CBr, and 8-bromoguanine, GBr, derivatives. The bromination products formed in molar ratio close to T(OH)Br/CBr/GBr ) 0.2:1:0.23 and serve as internal electron scavengers on γ-irradiation. Paramagnetic products that result from electron scavenging in DNA by T(OH)Br and CBr units at 77 K have been identified by ESR as the 6-hydroxy-5,6-dihydrothymin-5-yl (TOH•) radical and the 5-bromocytosine σ* radical anion, CBr•-. Our quantitative estimates show that electron scavenging by T(OH)Br in brominedoped DNA is over an order of magnitude more efficient than the more abundant CBr traps. This indicates that there is a high probability the electron survives encounters with the planar CBr traps through either transmission or reflection. The yields of electron scavenging by T(OH)Br moieties have been treated quantitatively considering the scavenging process as a competition between diffusion of electrons to T(OH)Br traps and their fixation on cytosines in the form of protonated radical anions. A mean displacement of the electron from its entry point evaluated using this model is about 11 bases at 77 K. After trapping at 77 K no further migration takes place until annealing to temperatures near 150 K and above. At these temperatures electron migration is activated and migration distances are found to increase with temperature likely through a hopping mechanism.
Introduction Electron transfer within DNA is an intriguing problem that has attracted significant attention during the past decade.1-18 The characteristic of base stacking in B-DNA with its linked π-orbital system closely resembles the crystal structure of organic semiconductors such as phthalocyanines.19 This similarity led to the concept that native DNA might behave as a one-dimensional semiconductor or even as a conductor capable of transporting charge carriers for long distances.20-23 From a radiobiological point of view this might enable the DNA strand to disperse radiation damage and resist the production of locally multiply damaged sites. Such sites are proposed as lesions that eventually lead to cell death.24 There are several stages of electron migration in DNA.25 In this work we consider the migration of thermalized excess electrons to sites of high electron affinity: thymine, cytosine, or dopants.18,26-31 The DNA strand can be considered as a onedimensional array of energetically near equivalent trapping sites for the electron. Trap-to-trap tunneling of the electron could occur in either direction, resembling a diffusion process.14 There is also a competitive chemical process that involves protonation of cytosine radical anions at a nitrogen atom (N3).28,32-36 This results in the stabilization of the electron in a deeper trap. However, the protonation at nitrogen is reversible34 and a somewhat higher activation energy is required to resume the †
Oakland University. Bowman Gray School of Medicine of Wake Forest University. X Abstract published in AdVance ACS Abstracts, February 1, 1997. ‡
S1089-5647(96)03094-5 CCC: $14.00
migration process. There is an additional protonation pathway that involves the C6 position in thymine radical anion that comes into play at higher temperatures. It irreversibly transforms thymine radical anions into 5,6-dihydrothymin-5-yl radicals, TH•, and ultimately quenches electron migration in DNA.37 Several techniques employed to investigate electron migration in DNA had resulted in quite different and somewhat conflicting results. The variety of reported values in part originates from the different experimental procedures employed. In the present paper we will consider a displacement of the electron from its “entry point” as a result of a random walk process. Experimentally such conditions have probably been realized in some experiments with intercalated or chemically incorporated electron traps.4,8,9,17 Photoinduced electron transfer3,5,10,11,13 presents a very different situation because (a) the electron movement is directional and (b) it implies tunneling between donor and acceptor sites without intermediate localization of the electron on any DNA base. Migration distances obtained using radiationinduced luminescence techniques1,2 are likely measurements of mean geminate electron-hole recombination distances reflecting both electrostatic interactions and tunneling. Long-distance tunneling may also be involved in electron trapping by a positively charged intercalate7 at low temperatures, when dielectric relaxation of the medium is slow. Low-temperature microwave conductivity measurements have recently been shown to reflect electron mobility in the water mantle of DNA rather than in the DNA strand itself.16 In the present paper we present a diffusion-based model of competitive electron trapping in DNA by both intrinsic and © 1997 American Chemical Society
Competitive Electron Scavenging artificially incorporated electron traps. We consider the fate of thermalized electrons that have escaped primary recombination with their parent holes. The earlier stage of electronhole separation in DNA has been considered previously using model distribution functions to describe the initial separation distances.2 A mathematical expression that describes the yield of electron scavenging in DNA has been reported by Cullis et al.7 However, its origin was not explained. Experimental Section In the present paper the DNA structure has been modified chemically to introduce an electron trap directly into the base sequence. Aqueous bromine is well-known to react readily with thymine double bonds, forming electron-scavenging bromohydrine functions.38 In a typical procedure employed in this work, bromine vapor slowly evolving from 0.1 M bromine aqueous solution was absorbed by 5 mL of ice-cold continuously stirred aqueous solution containing 20 mg of native salmon testes DNA (Sigma) per milliliter. This maintained a very low steady state concentration of bromine in solution. The amount of bromine employed was in accord with the desired bromine-tobase ratio and never exceeded 1 bromine per 4 DNA bases. Thus bromine was never present in excess with respect to DNA. The reaction was conducted in a sealed all-glass ice-cooled vessel and required several hours to complete. Immediately after the bromination the solutions were freeze-dried, yielding a fibrous mass used in further experiments. The same procedure was applied to brominate polyC‚polyG double-stranded polymer and individual nucleotides. The DNA samples brominated at the highest level were tested for DNA integrity using optical absorption spectroscopy and ethidium bromide as an indicator. Ethidium cation is known to have much higher affinity for double-stranded DNA than for single-stranded DNA. Intercalation in DNA results in a pronounced red shift of the ethidium absorption spectrum in the visible, which has been used as an indicator of binding efficiency. The UV-vis spectrum was taken of a solution containing 1.3 mg mL-1 of brominated DNA with ca. 10 µM ethidium bromide in phosphate buffer (pH ) 7). A solution containing native double-stranded DNA served as a control sample. Another control sample contained ethidium bromide alone in the buffer. We found that bromination slightly changed the absorption background likely due to an increased light scattering with no substantial effect on the absorption maximum position (520 nm) or intensity. On this basis we conclude that DNA remains largely in its double-stranded form even at the highest bromination levels. We find that the bromination of thymidine 5′-monophosphate (TMP) quantitatively produces the mixture of cis- and transisomers of 5-bromo-6-hydroxy-5,6-dihydrothymine 5′-monophosphates (T(OH)Br), identified by NMR (D2O, δ(CH3) ) 1.941 ppm, δ(6-H) ) 5.599 ppm, 60%, and δ(CH3) ) 1.948 ppm, δ(6-H) ) 5.483 ppm, 40%). The 2′-deoxycytidine 5′monophosphate has been shown by HPLC to produce mostly 5-bromo-2′-deoxycytidine 5′-monophosphate (CBr, 80%) and minor amounts of hydrolysis and deamination products. Bromination of purine nucleotides (dAMP, dGMP) was found to be less selective. The most abundant product in both cases has been identified as an 8-bromo derivative formed with about 60% yield. There were a number of other unidentified products, including some causing a bright yellow or orange coloration of the reaction mixtures. The structures and abbreviations referred to further in the text are depicted below. The bromination products formed in DNA were analyzed using gas chromatography/mass spectrometry (GC/MS) techniques. Due to the necessity for an acid hydrolysis of the DNA
J. Phys. Chem. B, Vol. 101, No. 8, 1997 1461 TABLE 1: GC/MS Product Analysis of Bromine-Doped DNA bromine content, expecteda
bromine content, founda
T(OH)Br, nmol/mg DNA
GBr, nmol/ mg DNA
CBr, nmol/ mg DNA
4 8 12 16 20 24 28
3.9 7.2 10.3 15.2 25.6 30.0 44.1
160 32 34 22 19 5 7
173 70 62 45 11 16 5
308 255 155 105 73 66 48
a Number of DNA bases per one covalently bound bromine. Expected values are based on base-to-bromine molar ratios employed during bromination of DNA.
samples in preparation for GC/MS analysis,39 a number of the brominated bases were quantitated based on their corresponding hydrolysis products.40,41 Briefly, the brominated DNA samples were divided into two sample sets. The first set was treated with a 50% aqueous pyridine solution at 60 °C for 1 h, lyophilized, and then hydrolyzed in 88% formic acid at 140 °C for 90 min. When applied to brominated thymidine 5′monophosphate, the treatment was found to convert T(OH)Br to 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol). The second set of brominated DNA samples was hydrolyzed only in 88% formic acid. This treatment converted CBr to 5-bromocytosine and some minor hydrolysis products (e.g. 5-hydroxyuracil and 5-hydroxycytosine) and the 8-bromo derivatives of dAMP and dGMP to 8-hydroxyadenine and 8-oxoguanine, respectively. The hydrolysis products were then trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and analyzed by GC/MS.39 Results of our analytical analyses shown in Table 1 indicate that the overall bromination level of the DNA corresponds to the nearly quantitative reaction of bromine with DNA bases. The predominant product was found to be CBr along with lessor amounts of T(OH)Br and GBr. The ratio between two latter products as well as their ratio to CBr showed some variation from sample to sample. However, except for one sample at the highest bromination level, there is no indication this variation is related to the bromination level at least in the case of T(OH)Br. On this basis the variations were treated as a random deviation from the mean product ratio calculated to be T(OH)Br/CBr/GBr ≈ 0.20:1:0.23 for the whole series of brominated
1462 J. Phys. Chem. B, Vol. 101, No. 8, 1997
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samples. The following analysis of experimental data will be based on this ratio (one out of 7.12 brominated bases assumed to be T(OH)Br) and experimentally measured overall bromination levels given in the second column of Table 1. The only exception is the sample doped at the highest level, where each fourth brominated base is T(OH)Br. No appreciable bromination of adenine was detected by GC/MS. Typically the samples were irradiated and studied in the form of frozen cylindrical plugs prepared from 20 mg of brominated DNA in about 80 µL of water or 10 mg of brominated nucleotides in 0.1 mL of water in oxygen-free atmosphere. For some experiments DNA and polyC‚polyG samples were also humidified over a saturated solution of potassium chloride in D2O that provides a hydration level of approximately 14 water molecules per nucleotide. The typical irradiation dose was about 20 kGy. Irradiation and ESR measurements were performed as described earlier.42 The spectra were analyzed using a set of experimental benchmark spectra within a linear least squares approach also described in our earlier publication.43 Results Identification of Electron-Trapping Sites in BromineDoped DNA. Figure 1A shows the spectrum of γ-irradiated hydrated bromine-doped DNA containing approximately 1 covalently bound bromine per 4 bases. The level of hydration employed (14 waters/nucleotide) is well below the level where a separate crystalline ice phase begins to form, avoiding the production of hydroxyl radicals trapped in the ice phase.42,44-48 However, it has been shown recently that a small concentration of hydroxyl radicals can be trapped in a glassy hydration layer around the DNA.48 These species are responsible for the broad low-field resonance that quickly disappears on annealing to 120 K. The ESR spectrum of γ-irradiated native DNA is well-known to be a poorly resolved doublet formed by superposition of the spectra of one-electron-reduced pyrimidines (mostly cytosine)18,26,27,30,31,49,50 and one-electron-oxidized purines (mostly guanine).30,51-54 There are also broad almost structureless wings believed to belong in part to neutral sugar radicals.55 The spectrum of bromine-doped DNA clearly displays two extra features not observed in native DNA. One of them, also found in brominated polyC‚polyG (Figure 1B) and frozen aqueous solutions of 5-bromo-2′-deoxycytidine 5′-monophosphate (Figure 1C), is assigned to the σ* radical anion CBr•-. These species show a large anisotropic bromine hyperfine coupling (a|(Br) ≈ 110 G) reported earlier for the 5-bromouracil σ* radical anion in low-temperature glasses56,57 and 5-bromouracilsubstituted DNA.58 We found no evidence for the production of π* radical anions of CBr, although such species were reported to exist in the case of 5-bromouracil.58-60 The other feature is identical to the spectrum found for γ-irradiated frozen aqueous solution of T(OH)Br (Figure 1D) and assigned to the 6-hydroxy5,6-dihydrothymidin-5-yl monophosphate radical TOH• a(CH3) ) 23 G and a(C6-H) ) 5 G proton hyperfine couplings.38 Previous work has shown that the TOH• radical is the normal product of dissociative electron capture by T(OH)Br units and this is found to extend to T(OH)Br in brominated DNA.38 In the case of 5-halogenated uracils this process is known to involve the intermediate formation of relatively long-lived radical anions that can be trapped at low temperatures.56-60 Our result indicates that this behavior is typical also for 5-bromocytosine derivatives as well. Moreover, the CBr•- species generated in both brominated DNA and polyC‚polyG show no tendency to dissociate on annealing to at least 200 K. The yield of CBr•- species was estimated in DNA containing about 1 covalently bound bromine per 4 nucleotides. The
Figure 1. (A) ESR spectrum of γ-irradiated bromine-doped DNA humidified over saturated KCl solution in D2O (hydration level about 14 waters per nucleotide). Bromine content is about 1 atom per 4 nucleotides. The spectrum was taken at 77 K after annealing the sample at 150 K for 10 min. Double and single arrows indicate the features of CBr•- and TOH• radicals, respectively. Three markers in the spectrum are Fremy salt resonances separated by 13.09 G and centered at g ) 2.0056. (B) The spectrum of bromine-doped polyC‚polyG doublestranded polymer treated in a similar manner and showing the features of CBr•- species. Bromine content is about 1 atom per 8 cytosine bases. (C) The spectrum of γ-irradiated frozen solution of 5-bromo-2′deoxycytidine monophosphate in D2O (100 mg/mL) taken at 77 K after annealing at 150 K. The most abundant species in the spectrum is the CBr•- radical anion, whose features are indicated by the arrows. (D) ESR spectrum of TOH• radicals obtained by irradiation of 5-bromo6-hydroxy-5,6-dihydrothymidine monophosphate in frozen D2O at 150 K. Arrows indicate the features clearly observable in brominated DNA.
spectrum of the same species produced by irradiation of the 5-bromocytidine monophosphate was taken as a benchmark spectrum. At 77 K CBr•- species constitute not more than 3% of all one-electron-reduced products. Annealing noticeably increases the contribution of CBr•- species in the spectra, but their yield has never been found to exceed 15% of the yield of TOH• species. Even in bromine-doped polyC‚polyG polymer where the concentration of CBr units accounts for 25% of the total number of cytosine bases, only 50% of electrons were found to be scavenged at CBr sites. This indicates that CBr units at low temperature are much less efficient electron scavengers than T(OH)Br moieties; we estimate that CBr is only about 3 times more effective than cytosine itself. At this time we have no evidence that GBr acts as a significant competitive electron trap in bromine-doped DNA. The spectrum produced by γ-irradiation of brominated 2′-deoxyguanosine monophosphate is a singlet that would be difficult to recognize in irradiated DNA. However, guanine is not competitive with cytosine for the electron, and GBr should be similarly noncompetitive with CBr. Further we note that CBr is shown in this work to be a noncompetitive electron scavenger relative to T(OH)Br in DNA at low temperatures. GBr is therefore not
Competitive Electron Scavenging
Figure 2. ESR spectra of bromine-doped (25 mol % Br) and natural DNA in water (25 wt % DNA) taken on annealing from 90 to 200 K. The spectra for each sample are taken at the same gain except for the spectra at 200 K. Growth of TOH• signal in brominated DNA on annealing from 90 to 150 K indicates the existance of thermally activated electron transfer from protonated cytosine radical anions to T(OH)Br sites. In natural DNA the migration finally results in the formation of TH• radicals. Yields of TOH• and TH• radicals shown are calculated relative to the initial concentration of DNA-based radicals at 90 K.
expected to compete effectively for electrons with T(OH)Br when both are present in comparable amounts in DNA. The Yields of Electron Scavenging by T(OH)Br Units in Bromine-Doped DNA. Seven bromine-doped wet DNA samples with the approximate bromine content ranging from 0.26 to 0.023 dopant atom per DNA base were irradiated at 77 K to the dose 20 kGy and measured at 90 K and after annealing at 120, 150, 170, and 200 K. Figure 2 shows the spectra of the sample at the highest doping level taken at several temperatures. The yield of TOH• varies from about 27% at 90 K to 40% at 150 K of the total original signal intensity. Figure 3 compares the spectra at several doping levels taken at 120 and 200 K. Annealing at 120 K has little effect on the spectrum of DNAassociated radicals but removes hydroxyl radicals trapped in the ice phase. The major event at 200 K is formation of TH• radicals in natural DNA; this process is strongly suppressed in brominated DNA samples. The wings of the spectra display predominant features of TOH• and TH• radicals, superimposed over a low-intensity structureless tail. These three components were quantified using the least squares deconvolution procedure and a set of benchmark spectra representing the individual “species”. The underlying background signal was taken from the ESR spectrum of wet native DNA irradiated at 77 K to the same dose. The TOH• benchmark spectra were taken in irradiated frozen aqueous solutions of T(OH)Br (100 mg mL-1) after annealing at 120, 150, 170, and 200 K and employed in the analyses of DNA spectra of the same temperature. The TH• benchmark spectrum was the same as employed in previous work.61 It is found (Figure 2) that TOH• radicals remain stable at temperatures up to 150 K, but further temperature increases result in partial loss in concentration. For this reason the data obtained above 170 K were excluded from quantitative treatment.
J. Phys. Chem. B, Vol. 101, No. 8, 1997 1463
Figure 3. ESR spectra taken at 120 and 200 K at different bromination levels to show the dopant effect on the yields of TOH• and TH• radicals in brominated DNA. The integrated intensities of the spectra taken at one temperature are normalized to the same value. Even low bromination levels substantially suppress the production of TH• radicals in DNA at 200 K, which indicates the involvement of thermally activated electron migration in the reduction of thymine.
Figure 4. Experimental scavenging yields by T(OH)Br traps in brominated DNA (TOH• yields normalized to the overall yield of oneelectron-reduced products in DNA) plotted vs an average number of bases between the traps (N). Note that scavenger concentrations decrease with increasing N. Solid curves are calculated using the diffusioncontrolled model with one adjustable parameter, R. Mean electron migration distances used in these calculations are specified. The middle solid curve shows the least squares fit to the experimental data set with the first point excluded. This fit is identical for the models with either one (R) or two (R,β) adjustable parameters and gives our best estimate of n. The dotted line represents the least squares fit to the full data set using the model with one adjustable parameter, R. The dashed line represents the least squares fit to the full data set using the twoparameter model.
To obtain the yield of electron scavenging (Rsc), the yields of TOH• radicals were normalized to the yield of radical anions in original DNA, estimated to be about 60% of the total radical signal at 90 K.18 The experimental yields measured at 90 K are plotted in Figure 4 as a function of the number of bases per one T(OH)Br unit calculated as described in the Experimental Section. The measured scavenging yields at 90 K indicate that T(OH)Br intercepts from 10% to 50% of all electrons on going from the lowest to the highest doping level. In Figure 5 the
1464 J. Phys. Chem. B, Vol. 101, No. 8, 1997
Figure 5. Electron-scavenging yields plotted vs the reciprocal separation distance (1/N) between the traps. Temperatures of annealing and migration distances (n) calculated from the linear portions of the plots are specified. The data at 90 and 120 K show no significant difference and are treated as one set. The data at 90, 120, and 150 K are based on direct measurements of TOH• yields, while the data at 200 K are calculated from the fractional reduction in the yield of TH• radicals at this temperature.
same data are plotted together with the yields measured after annealing at higher temperatures. The data are plotted in reciprocal coordinates to illustrate expected hyperbolic dependencies of the yields on the distance between T(OH)Br units at low concentrations of the scavenger (see the Discussion section). Slow annealing of γ-irradiated hydrated DNA above 170 K typically produces a significant amount of TH• radicals whose yield maximizes within the 195-205 K temperature range. At low irradiation doses the yield of TH• can reach more than 30% of the initial radical concentration.61 We found it close to 27%, which is consistent with the expected value of about 25% for 20 kGy.61 Bromine doping suppresses the TH• yields far more than it suppresses the yields of their anionic precursors, i.e. cytosine and thymine electron adducts. Even the presence of one covalently bound bromine per 44 bases reduces the TH• yield by about 50%. This is clearly a result of competition between unaltered thymines and brominated bases for the electrons on thermal detrapping from the cytosine-located temporary traps on annealing. Unfortunately, the TOH• radical cannot be used as an indicator of electron-scavenging efficiency because it lacks thermal stability over 160 K. An attempt was made to deduce the scavenging yields from the effect of bromine doping on TH• yields. To obtain such an estimate, the differences in TH• yields in native and brominated DNA must be normalized to the TH• yield in native DNA. The scavenging yields calculated in this way are plotted in Figure 5 together with the data obtained directly from TOH• yields at lower temperatures. Some problems associated with this treatment are discussed in the following section. Discussion Nature of the Trap and Scavenging Efficiency. An unexpected finding of the present study is a striking difference between T(OH)Br and CBr entities in their electron-scavenging capabilities. If both reacted with the migrating electron on every encounter, the yields of scavenging products (which are TOH• and CBr•- radicals, respectively) would reflect the ratio between the scavengers in DNA, which is about 1:5. In fact the relative yields are approximately 6:1 in favor of TOH• production and can only be explained assuming partial “transparency” of CBr traps to the migrating electron. This suggests domination of kinetic- over diffusion-controlled electron scavenging for 5-bromocytosine; however, for T(OH)Br we believe the system approaches a diffusion-controlled scavenging.
Razskazovskii et al.
Figure 6. In-plane views of space-filling models for 5-bromocytosine and 5-bromo-6-hydroxy-5,6-dihydrothymine molecules. The structures are optimized using a simple molecular mechanics approach.
There are at least two likely reasons why these bromo derivatives behave so differently in DNA. First, the long lifetime of CBr•- anion may allow the scavenging to be reversible, while T(OH)Br traps likely act as an irreversible sink for the electrons, forming an unstable electron attachment intermediate, resulting in TOH•. A consideration of the structures of CBr and T(OH)Br suggests the second reason. In Figure 6 we show in-plane views of these two species with R ) H found after a simple molecular mechanics optimization. A bromine atom has almost the same van der Waals radius as a methyl group and is expected to cause little disturbance in DNA structure on substitution for in-plane hydrogens in DNA bases. Thus formation of CBr from cytosine and GBr from guanine leaves both the π system and stacking within the DNA strand largely undisturbed. This is definitely not the case for thymine bromination, where saturation of the double bond creates a system with out-of-plane arrangement of both bromine and OH groups. Figure 6 shows the trans-T(OH)Br isomer, which is likely more abundant. The cis-isomer may also be present and would be nearly as disruptive. The cis or trans structures clearly will create a break in the π stacking and induce a bend or kink in the overall DNA configuration. These structural alterations will likely hinder passage of the electron past the T(OH)Br center. Thus the assumption that these T(OH)Br centers efficiently terminate electron migration within DNA is reasonable. Scavenging Yields and Electron Migration Distances. The mathematical treatment of experimental data proposed below is based on the following simplifications. 1. The DNA strand is a uniform one-dimensional continuum containing evenly distributed electron scavengers. 2. Electrons are injected into the strand anywhere with equal probabilities. 3. Electron migration in DNA can be treated as a onedimensional diffusion. The diffusion coefficient is related to an electron-hopping distance d and an average lifetime at one location τd through the classic expression
D ) d2/2τd
(1)
The average lifetime τd is also a reciprocal rate constant of tunneling between neighboring sites ket. 4. The diffusion process competes with stabilization of the injected electron in the form of protonated cytosine radical anion.
Competitive Electron Scavenging
J. Phys. Chem. B, Vol. 101, No. 8, 1997 1465
The protonation is characterized by a rate constant (kp) or a characteristic lifetime, τp ) 1/kp. Such stabilization is assumed to be equally probable everywhere within the strand. The electron can also reach the scavenger and react with it with the rate constant ksc ) 1/τsc, which is in fact an electron transfer rate from the neighboring base to the scavenger. 5. T(OH)Br is the only scavenger of the electron. Others including CBr and GBr are assumed to be indistinguishable from unaltered DNA bases. 6. Electrostatic interactions and recombination processes involving electrons and radiolytically generated holes in DNA are neglected. Thus we are considering the fate of only those electrons that escaped geminate recombination and no longer feel the electric field of the parent holes. 7. Interstrand electron transfer is considered to be noncompetitive with electron scavenging within the same strand. As a result, the mean separation between the trapping sites in the strand is considered to be equal to the number of bases per one trap. The solution of a steady state diffusion problem for the situation described above is given in the Appendix. The general solution for the scavenging yield Rsc can be presented as a function of the variable N, being the number of bases per dopant atom:
Rsc )
β RN(R + β coth(RN))
(2)
It also depends on two dimensionless parameters R ) (τd/2τp)1/2 and β ) τd/τsc. The parameter R characterizes the relative rate of diffusion vs protonation process and thus characterizes the migration process itself regardless of the presence of the scavenger. This parameter is related to the number of diffusion movements made by the electron per one protonation event that can be expressed as the τp/τd ratio. We consider it to be equivalent to the mean number of hops made by the electron between entering the strand and its trapping by protonation. The square root of this number gives the mean displacement of the electron from its entry point ∆x, which we define as a migration distance measured in (the number of) base pairs. Thus the relationship between the parameter R and the migration distance ∆x is just ∆x ) 2-1/2R-1. The parameter β defines the relative efficiency of the diffusion vs scavenging process. In the case of much faster scavenging it can easily become large enough to satisfy the condition β coth(RN) . R for all reasonable values of N. In this case expression 2 reduces to a simpler one that no longer includes the parameter β.
Rsc )
1 tanh(RN) RN
(3)
The scavenging now occurs in a diffusion-controlled regime. This means that every electron reaching the scavenger is trapped with unit probability. The nonlinear least squares fit of experimental TOH• yields using the general formula 2 with two adjustable parameters is shown in Figure 4 with a dashed line. It results in R ) 0.0065 and β ) 0.74 × 10-3 values for the parameters of the model. In terms of electron migration distances ∆x this would mean that electrons in DNA become localized about 109 bases away from their entry point. However, the β value implies a scavenging rate more than 1000 times slower than the diffusion rate. This is not very reasonable. An alternative treatment presumes a diffusion-controlled scavenging, implying that the electron is transferred much faster from the DNA base to the scavenger than just from one base to another. Fitting the full
set of the data using the reduced formula 3 with one adjustable parameter gives R ) 0.10, which corresponds to the ∆x ) 7 bases. However, the quality of this fit shown in Figure 4 with a dotted line is substantially worse than in the previous case. The discrepancy between the approaches disappears if the data representing the highest doping level are excluded from the treatment. In this case both formulas result in almost identical fits with R ) 0.064 (∆x ) 11 bases) coupled with a large β value (β g 10) in the first case. This fit is shown in Figure 4 with a solid line. We consider this result as a more realistic estimate of electron migration distance in DNA. The rationale for exclusion of the first point from the fitting procedure is that the change in DNA structure at high bromination levels affects its ability to serve as a medium for electron migration. Other curves in Figure 4 are theoretical ones calculated for illustration using formula 3 with ∆x values indicated near the curves. At large N when the values of either tanh(RN) or coth(RN) approach 1, both dependencies are expected to turn into the simple hyperbolic functions of N. The same type of dependence can be derived from a very simple consideration. If we assume that all electrons entering the strand within n base pairs from the T(OH)Br entities are scavenged while all others are stabilized as radical anions, the scavenging yield is simply expressed as Rsc ) 2nscN-1. An alternative asymptotic expression that can be derived from (3) at large N’s is Rsc) (RN)-1. Thus there is a very simple relation between the parameter R of the diffusioncontrolled model and the effective scavenging range n, i.e., nsc ) 0.5R-1. This also leads to the relationship between the effective scavenging range and the mean displacement introduced above: ∆x ) 21/2nsc. The effective scavenging ranges can also be calculated from the scavenging yields obtained on annealing, as shown in Figure 5. Little increase in scavenging range is observed on annealing from 90 to 120 K; however, a substantial increase from 8.5 to 15 bases is found on further annealing to 150 K. This is a clear indication that thermal activation becomes an important component of the electron migration process in DNA on annealing to temperatures near 150 K. However, we note these effective scavenging ranges are no longer related to the parameters of the model, as it does not properly describe the processes that take place on annealing, which is likely a thermally activated hopping process. The ultimate fate of the electron in DNA on annealing to 200 K is the formation of TH•.61 Reduction of the yield of TH• with added scavenger can then be a measure of the migration distance. However, transformation of TH• yields measured at 200 K into electron-scavenging yields and, further, into the “scavenging range” is more problematic. The treatment proposed above implies that free radical concentration remains constant during the annealing procedure. This is a fairly good approximation at temperatures below 150 K, but it may not be valid at 200 K, where a substantial electron-hole recombination is involved. The role of CBr and GBr scavengers in the suppression of TH• production at 200 K is also unknown. These two are not competitive scavengers with respect to T(OH)Br at lower temperatures but are likely competitive scavengers with thymine itself. If the problems just discussed are neglected and T(OH)Br traps are assumed to be the only effective scavengers, then the “scavenging range” at 200 K obtained from a hyperbolic fit to the scavenging yields is about 75 bases, as shown in Figure 5. Given the above discussion, this value is likely an upper limit, and a more realistic estimate would lie between 20 and 40 bases. However, the pronounced effect of electron scavengers on the
1466 J. Phys. Chem. B, Vol. 101, No. 8, 1997 TH• yields clearly supports the earlier finding that thermally activated migration is involved in thymine reduction in DNA.61 The reliability of the estimates given in the present paper is based first of all on the reliability of T(OH)Br concentrations found in brominated DNA. Those are known with fairly large uncertainty and seem to be rather underestimated than overestimated. Fitting of the experimental TOH• yields assuming higher concentration of the scavenger would result in lower electron migration distances than reported. For this reason our value of 11 bases more likely overestimates rather than underestimates the electron migration distance in DNA at 90 K. Conclusion Our most reliable estimate of electron migration distance using T(OH)Br traps in hydrated DNA at 90 K is 11 bases. This distance reflects the migration range of thermalized electrons in the course of their transformation into new chemical entities, which are one-electron-reduced pyrimidines. This process is fast and likely almost activationless. It is fully completed at the temperature of irradiation (77 K). Additional debromination of T(OH)Br traps occurs only on annealing to 150 K. This is strong evidence for thermally activated electron transfer, which at these temperatures is a slow process, taking several minutes for full development of the scavenger radical signals. At higher temperatures C-protonation of one-electronreduced thymine forming TH•, which serves as the ultimate sink for the electron in native DNA, competes with the debromination of T(OH)Br in bromine-doped DNA. Our results suggest electron migration distances at these higher temperatures increase substantially perhaps beyond 30 bases. It should be emphasized that the migration distances or “scavenging ranges” reported in this paper are always interplays of electron hopping that sustains the migration and electron trapping that terminates it. If there were no trapping, the electron lifetime in DNA would be unlimited as well as its migration distance. For this reason our values should not be taken for the long-range electron-tunneling distances between donor and acceptor reported in some photochemical studies.3,5,10,11,13 Our results also raise a general question regarding the reliability of electron migration distances in DNA estimated using electron-scavenging techniques. There are two parameters that affect the results: the migration rate itself and the scavenging efficiency. To the best of our knowledge, this fact has never been emphasized in studies of the mobility of excess electrons in DNA. This point is not important when the scavenging is much faster than the migration process and reaction proceeds in a diffusion-controlled regime. However, if the scavenging process is kinetically controlled, ignoring the limited scavenging efficiency results in a large underestimate of real migration distances. Special care should be taken when using 5-halouracils (in particular, 5-bromouracil) as electron scavengers in DNA.8,9,62 The electron-scavenging properties of these compounds in DNA could be quite similar to those of 5-bromocytosine, which we find to have a scavenging efficiency only a few percent of that of T(OH)Br. Thus 5-bromocytosine is definitely not a diffusion-controlled scavenger in DNA, at least not at low temperatures. Other works have expressed intuitive expectations that scavenging yields in DNA would maximize at a certain distance between the scavengers on going from low to high concentrations of electron scavengers and then stay constant.8,9 This distance was taken as an estimate of electron migration distance in DNA. Our calculations do show flattening of the yield vs scavenger concentration dependencies at high concentrations of
Razskazovskii et al. the scavenger. However, even in diffusion-controlled situations there is no sharp feature on the curves that would allow recognition of the point where the flattening starts. In the case of kinetically controlled scavenging there may be no flattening at all even when the migration distance is relatively large. The dashed line shown in Figure 4 provides a good illustration to this statement. It shows no plateau although the migration distance in this is over 100 bases. Acknowledgment. We thank the National Cancer Institute of the National Institutes of Health (Grant RO1CA45424) and the Oakland University Research Excellence Program in Biotechnology for support of this work. Appendix A standard mathematical presentation of the steady state diffusion problem in a one-dimensional continuum containing evenly distributed electron sources and sinks is a differential equation for the probability density function W(x):63
d2W(x)
D
dx2
- kpW(x) + I ) 0
(4)
where D is a diffusion coefficient, kp is a protonation rate constant, and I is the rate of electron injection per unit length. The diffusion is considered to occur in a limited space confined between two dopant sites located at x ) 0 and x ) r. Scavenging is described by setting the boundary conditions at the dopant locations:63
dW(x) | ) kscδW(0) dx x)0
D
dW(x) | ) kscδW(r) dx x)r
-D
(5)
The left sides of eqs 5 represent the flux of the diffusing particles through the scavengers’ sites. The right sides are the reaction rates in a thin layer surrounding the scavenger expressed through the “volume” scavenging rate constant, ksc, and the thickness of the reaction layer, δ. Boundary conditions taken in the form (5) allow the diffusing particle either to pass through the trap, to be scavenged, or to be reflected back. If scavenging in DNA is treated as an electron transfer from the nearest base to the scavenger, the thickness of the reaction layer δ can be replaced by the interstack distance d. The yield of the electron trapping Rsc by the scavengers is calculated as the ratio of the total electron flux through the scavenger sites to the total number of electrons entering the fragment of DNA confined between these two sites. Taking into account the symmetry of the problem, the value to be found is
D dW(x) | Rsc ) 2 Ir dx x)0
(6)
Remembering that D ) d2/2τd, kp ) 1/τp, ksc ) 1/τsc, and δ ) d and introducing the new variables n ) x/d and W′(n) ) W(n) - Iτp, eq 4 may be presented in the form
d2W′(n) dn
2
- 4R2W′(n) ) 0
(7)
with the boundary conditions
dW′(n) | ) 2β(W′(0) + Iτp) dn n)0
(8)
Competitive Electron Scavenging
-
J. Phys. Chem. B, Vol. 101, No. 8, 1997 1467
dW′(n) | ) 2β(W′(N) + Iτp) dn n)N
and the expression for the scavenging yield
Rsc )
1 dW′(n) | tdNI dn n)0
(9)
The parameters R and β in eqs 7 and 8 are defined as (τd/ 2τp)1/2 and τsc/τd, respectively. The independent variable n is the distance measured by the number of bases, while the parameter N ) r/d is the number of bases located between two scavenging sites. A general solution for the homogeneous differential equation of second order (7) is
W′(n) ) C1 exp(2Rn) + C2 exp(-2Rn) where the coefficients C1 and C2 have to be found from the boundary conditions (8). After routine transformations the expression for the scavenging yield (9) can be obtained in the form given in the text:
Rsc )
β RN(R + β coth(RN))
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