Free radicals from X-irradiated 'dry' and hydrated lyophilized DNA as studied by electron spin resonance spectroscopy: analysis of spectral components between 77K and room temperature.

PURPOSE

To investigate the number, spectroscopic signatures and chemical structures of free radicals from X-irradiated lyophilized DNA (dry and equilibrated at 76% relative humidity) between 77 K and room temperature by electron spin resonance (ESR) spectroscopy.

MATERIALS AND METHODS

Samples were prepared by freeze drying DNA (sodium salt, salmon testes) in H2O or D2O and used as such ('dry' DNA) or after equilibration at 76% relative humidity. K3[Fe(CN)6] was co-lyophilized in some samples as an electron scavenger. X-irradiation was performed at 77 K (liquid nitrogen). Data acquisition was on a Bruker ESP 380 ESR-spectrometer (X-band, 9.5 GHz) and at high magnetic fields (245 GHz, Y-band; GHMFI, Grenoble, France). Data analysis involved computer treatment of spectra.

RESULTS

There were 12 different radical components isolated from DNA in four different conditions (dry and after equilibration at 76% relative humidity in either H2O or D2O) with the additional help of high magnetic field ESR and the use of K3[Fe(CN)6] as an electron scavenger. Several components were detected at 77 K and were found to be common for both hydration conditions, although their spectral shape varied considerably. These involved reduced thymine and cytosine bases, the oxidized guanine base, probably a C1'-located sugar radical, a thymine allyl radical and a secondary thymine H-addition radical. For the reduced cytosine base the amino-protonated form was observed in H2O samples, which was only partially exchanged in the D2O samples. At high water content another species, perhaps due to a sugar radical, contributes in addition even at low temperatures. All radical components anneal out with temperature, with only small secondary reactions taking place. A peroxy radical and a sharp singlet, probably due to the deprotonated radical cation from guanine, come into the balance together with the secondary thymine radical. At high doses, a further sugar radical (perhaps at the C3'-position) was detected in dry DNA. The relative yields of the isolated patterns were determined by precise reconstruction of the experimental spectra.

CONCLUSIONS

The comprehensive component delineation performed at 77 K and upon annealing to room temperature for lyophilized DNA showed a larger diversity and a higher variance of radicals at 77 K than discussed so far. Thermal annealing brings about only a few reactions to produce secondary species. Most components decay without paramagnetic successors.