Rate of OH Radical Induced Strand Break Formation in Single Stranded DNA under Anoxic Conditions. An Investigation in Aqueous Solutions Using Conductivity Methods

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Abstract
Single stranded DNA (ssDNA) in aqueous N2O-saturated solution was pulse-irradiated with electrons or irradiated under steady-state conditions with 60Co-.gamma. rays. The conductivity increase after irradiation was measured as a function of concentration, pH, temperature, metal cation content and additives. The conductivity increase was due to the release of associated counterions (Na+ or H+) as a result of the formation of chain breaks. At 28 mg l-1 DNA .apprx. 8 Na ions are liberated per 100 eV absorbed energy (G(Na+) = 8.3). On the basis of G value for single-strand breaks (ssb) of G (ssb) = 0.55, it is calculated that 8.3/0.55 = 15 Na ions per strand break are set free. The release of Na+ monitored by 23Na-NMR at pH 7 as a function of dose corresponds to that of the conductivity increase. The rate of the conductivity increase does not depend on dose/pulse (range 2-20 J kg-1) and shows at least 2 components. The rate constant of the fast and dominant component is constant above pH 8.5 (k = 38 s-1, 20.degree. C) and increases linearly with proton concentration below pH 7. Values of 13 kcal/mol for the activation energy and 5 .times. 1011 s-1 for the frequency factor were obtained at pH 7.3. Addition of p-benzoquinone (pBQ) increases the rate constant of the fast component proportionally to the concentration of pBQ. From these results the rate-determining step of the fast conductivity increase is concluded to be the splitting of DNA radicals. The pH dependence and the magnitude of the activation energy agree with results from low molecular weight model compounds for the C-4'' mechanism; this mechanism involves a heterolytic splitting of the phosphoric acid ester bond starting from the 4'' radical of DNA.