Oxidative damage is involved in the formation of free radicals, which can cause various diseases ranging from Alzheimer’s disease to cancer. One form of oxidative damage is DNA-protein crosslinking where proteins interact with lesions in the DNA. In DNA, this damage is observed primarily at guanine (G) because it is the most easily oxidized base. Therefore, we examined whether kale can inhibit oxidative DNA damage by using the flash-quench scheme. The flash-quench technique is a method that is used for guanine oxidation and can induce DNA-protein crosslinking. In the flash quench technique, the intercalator, Ru(phen)2dppz2+[phen = phenanthroline, dppz = dipyridophenazine], is excited with a laser and gives an electron to the quencher, Co(NH3)5Cl2+. The intercalator takes an electron from guanine, creating the guanine radical, which then reacts with protein. In our experiment, samples containing Ru(phen)2dppz2+, Co(NH3)5Cl2+, histone protein, PUC19 DNA and kale were irradiated with blue laser light for 0 to 120 seconds to induce guanine damage. The extent of crosslinking was determined by the gel shift assay where SDS was added to the samples to disrupt the noncovalent interactions between DNA and protein. Furthermore, the samples were compared with the control which substituted water for kale extract. Indeed, upon comparison, minimal DNA protein crosslinking was observed when kale was present in the samples. Specifically, our results showed as the irradiation time increased, the absorption of free DNA was low. The inhibition of oxidative DNA damage by kale was studied further by focusing on kaempferol, a component of kale. Likewise, the gel shift assay was performed on samples containing water and 200 µM kaempferol. Since kaempferol holds known antioxidative properties, then the inhibition of DNA oxidation at the guanine site was expected. In future work, emission spectroscopy will be used to determine whether the kaempferol affects the quenching process and to learn whether or not the antioxidants react with guanine radical directly.

Inhibition of oxidative DNA-protein crosslinking via an aqueous extract of kale

2019 Spring National Meeting

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