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

Abstract

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.