Glutathione is a small cysteine-containing peptide responsible for redox buffering in the cell. Here, we investigated whether it might be possible for glutathione, a strong reductant, to inhibit DNA damage (e.g. DNA-protein crosslinks) by engaging in redox repair of the 1-electron oxidized guanine base as formed by the flash-quench method. Samples containing Ru(phen)2dppz 2+ [phen = phenanthroline, dppz = dipyridophenazine], Co(NH3)5Cl2+, histone protein, calf thymus DNA were irradiated for 0-240 seconds in the absence or presence of glutathione with 442 nm light to effect guanine damage. In the chloroform extraction assay, we found that the 260 nm absorption of free DNA decreased with increasing irradiation time, consistent with crosslinking, and that the crosslinking plateaued at a higher value when the histone concentration was increased. In a separate experiment, the DNA samples were also subjected to ultrafiltration to remove the glutathione, which was then analyzed by reversed phase HPLC. Separation of the reduced and oxidized glutathione was observed by monitoring absorbance at the 220 nm wavelength. As irradiation time increased, the intensity of the oxidized glutathione peak increased, consistent with the redox repair mechanism described above. Furthermore, we compared the effect of reduced and oxidized glutathione in our samples via the gel shift assay and it was observed that the oxidized form had little to no effect on reducing the crosslinking, while the reduced form decreased crosslinking. Lastly, we investigated whether or not glutathione interfered with the quenching process by doing emission spectroscopy experiments. We found that glutathione only slightly interfered with the quenching of Ru(phen)2dppz 2+ , indicating that it most likely reacts with the guanine radical directly.

Characterization of glutathione inhibition of oxidative DNA-protein crosslinking

253rd National Meeting (2017)

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