Inhibition of Oxidative DNA‐protein Crosslinking Via an Aqueous Extract of Kale

Abstract

Oxidative damage is involved in the formation of free radicals, and if deregulated, can ultimately facilitate a variety of diseases ranging from Alzheimer’s disease to cancer. DNA‐protein crosslinking is a recognized form of oxidative stress that involves proteins interacting with radical‐induced lesions in the DNA molecule. In DNA, this damage is observed primarily at guanine (G) since it is the most easily oxidized base. Studies on oxidative stress show that substances – the most familiar being kale and spinach – high on antioxidants aid in protection from the excessive oxidizing effects of free radical species. The exact mechanism of inhibition has been hypothesized as a redox mechanism between the antioxidant and the radical. Therefore, the primary counteraction used to observe this mechanism and to reverse oxidative DNA‐protein crosslinking in this experiment was kale.The flash‐quench technique is a method used for guanine oxidation and can induce DNA‐protein crosslinking. 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 then takes an electron from guanine, creating the guanine radical, which then reacts with protein. Samples containing Ru(phen)2dppz2+, Co(NH3)5Cl2+, histone protein, PUC19 DNA and kale were irradiated with blue laser light for 0 to 140 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 the DNA and protein. Furthermore, the samples were compared with the control which substituted water for the kale extract. 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 two different concentrations – 50 μM and 200 μM – of the antioxidant. The interaction between kaempferol and the guanine radical was then displayed through transient absorption spectroscopy. Since kaempferol holds known antioxidative properties, the inhibition of DNA oxidation at the guanine site was also expected. Indeed, upon comparison, minimal DNA protein crosslinking was observed when kale or kaempferol were present in the samples. Specifically, as the irradiation time increased, the absorption of free DNA was low, assuming that a redox mechanism between the antioxidant and the guanine radical occurred.Future directions include determining the lowest concentration at which kaempferol is active, assessing the effects of additional antioxidants from kale, and examining a pure sample of kaempferol via high performance liquid chromatography (HPLC).Support or Funding InformationSupported and Funded by:Loring‐Denault Endowed Chair in Chemistry, Dr. Eleanor Siebert, and Professor Emeritus