The 2-deoxyribose degradation assay

This method is based on the determination of malonaldehyde, an oxidation product of 2-deoxyribose, by measurement of the colored condensation product with TBA at 532 nm (Andrade et al., 2006). In typical reactions, hydroxyl radical was generated by Fe(III)-citrate (1:1) or Fe(III)-EDTA (1:1) plus ascorbate in buffered media (20 mM phosphate, pH 7.2) (see reactions 1–4). After 10 min pre-incubating persimmon extracts with the Fe(III) complex (using citrate or EDTA as the ligand) and 2-deoxyribose, reactions were initiated by ascorbate addition, carried out for 30 min at room temperature (24–25 °C) and stopped by the addition 0.5 mL 4% (v/v) phosphoric acid followed by 0.5 mL TBA solution. Solutions were incubated for 15 min in boiling water bath and allowed to cool to room temperature before absorbance measurement. Each experimental result was corrected for its correspondent blank reaction (called “time zero” blank) to eliminate the background interference. In the “time zero” blank, ascorbate was added to the media after phosphoric acid and TBA addition (Andrade et al., 2006). This also discounts for 2-DR oxidation by Fe(III) in the analytical phase of the assay (Genaro-Mattos et al., 2009).