Presentation Title

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

Faculty Mentor

Eric Stemp

Start Date

23-11-2019 1:00 PM

End Date

23-11-2019 1:15 PM

Location

Markstein 211

Session

oral 3

Type of Presentation

Oral Talk

Subject Area

behavioral_social_sciences

Abstract

Oxidative damage is involved in the formation of free radicals, and if deregulated, facilitates various 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. In DNA, this damage is observed primarily at guanine (G), the most easily oxidized base. Studies on oxidative stress show that substances high in antioxidants aid in protection from the oxidizing effects of free radical species. Therefore, the primary counteraction used to reverse oxidative DNA-protein crosslinking was kale.

The flash-quench technique is a method that involves inducing DNA-protein crosslinking and guanine oxidation. Here, the Ru(phen)2dppz2+[phen = phenanthroline, dppz = dipyridophenazine] intercalator is excited with a blue laser light and reduces the Co(NH3)5Cl2+ quencher. The intercalator is then reduced by the guanine, creating the guanine radical, which can react with protein. Samples containing the intercalator, quencher, histone protein, PUC19 DNA, and kale were irradiated for differing times 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 DNA-protein noncovalent interactions. These samples were compared with the control, which substituted water for the kale extract. This inhibition process was studied further by focusing on kaempferol, a component of kale, in different concentrations.

Additionally, the interaction between kaempferol and the guanine radical was displayed through transient absorption spectroscopy. Since kaempferol holds known antioxidative properties, then the inhibition of DNA oxidation at the guanine site was expected. Upon comparison, minimal DNA protein crosslinking was observed when antioxidant was present in the samples. Specifically, as the irradiation time increased, the absorption of free DNA was low.

Future directions include assessing the effects of additional antioxidants from kale and examining a pure sample of kaempferol via high-performance liquid chromatography (HPLC).

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Nov 23rd, 1:00 PM Nov 23rd, 1:15 PM

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

Markstein 211

Oxidative damage is involved in the formation of free radicals, and if deregulated, facilitates various 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. In DNA, this damage is observed primarily at guanine (G), the most easily oxidized base. Studies on oxidative stress show that substances high in antioxidants aid in protection from the oxidizing effects of free radical species. Therefore, the primary counteraction used to reverse oxidative DNA-protein crosslinking was kale.

The flash-quench technique is a method that involves inducing DNA-protein crosslinking and guanine oxidation. Here, the Ru(phen)2dppz2+[phen = phenanthroline, dppz = dipyridophenazine] intercalator is excited with a blue laser light and reduces the Co(NH3)5Cl2+ quencher. The intercalator is then reduced by the guanine, creating the guanine radical, which can react with protein. Samples containing the intercalator, quencher, histone protein, PUC19 DNA, and kale were irradiated for differing times 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 DNA-protein noncovalent interactions. These samples were compared with the control, which substituted water for the kale extract. This inhibition process was studied further by focusing on kaempferol, a component of kale, in different concentrations.

Additionally, the interaction between kaempferol and the guanine radical was displayed through transient absorption spectroscopy. Since kaempferol holds known antioxidative properties, then the inhibition of DNA oxidation at the guanine site was expected. Upon comparison, minimal DNA protein crosslinking was observed when antioxidant was present in the samples. Specifically, as the irradiation time increased, the absorption of free DNA was low.

Future directions include assessing the effects of additional antioxidants from kale and examining a pure sample of kaempferol via high-performance liquid chromatography (HPLC).