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 chemi cal structures of free radicals from X-irradiated lyophilized DNA (dry and equilibrated at 76% relative humidity) between 77 K and room temp erature by electron spin resonance (ESR) spectroscopy. Materials and m ethods: Samples were prepared by freeze drying DNA (sodium salt, salmo n testes) in H2O or D2O and used as such ('dry' DNA) or after equilibr ation at 76% relative humidity. K-3[Fe(CN)(6)] was co-lyophilized in s ome samples as an electron scavenger. X-irradiation was performed at 7 7 K (liquid nitrogen). Data acquisition was on a Bruker ESP 380 ESR-sp ectrometer (X-band, 9.5 GHz) and at high magnetic fields (245 GHz, Y-b and; GHMFL, Grenoble, France). Data analysis involved computer treatme nt of spectra. Results: There were 12 different radical components iso lated from DNA in four different conditions (dry and after equilibrati on at 76% relative humidity in either H2O or D2O) with the additional help of high magnetic field ESR and the use of K-3[Fe(CN)(6)] as an el ectron scavenger. Several components were detected at 77 K and were fo und to be common for both hydration conditions, although their spectra l shape varied considerably. These involved reduced thymine and cytosi ne bases, the oxidized guanine base, probably a C1'-located sugar radi cal, a thymine allyl radical and a secondary thymine H-addition radica l. For the reduced cytosine base the amino-protonated form was observe d in H2O samples, which was only partially exchanged in the D2O sample s. Al high water content another species, perhaps due to a sugar radic al, contributes in addition even at low temperatures. All radical comp onents anneal out with temperature, with only small secondary reaction s taking place. A peroxy radical and a sharp singlet, probably due to the deprotonated radical cation from guanine, come into the balance to gether with the secondary thymine radical. At high doses, a further su gar radical (perhaps at the C3'-position) was detected in dry DNA. The relative yields of the isolated patterns were determined by precise r econstruction of the experimental spectra. Conclusions: The comprehens ive component delineation performed at 77 K and upon annealing to room temperature for lyophilized DNA showed a larger diversity and a highe r variance of radicals at 77 K than discussed so far. Thermal annealin g brings about only a few reactions to produce secondary species. Most components decay without paramagnetic successors.