Fig. 3

The antioxidative activity of uuferritin in vitro. a The DNA binding activity of uuferritin is promoted by either Fe²⁺ or H₂O₂. The DNA binding activity of uuferritin was analyzed by the capacity to retard the migration of supercoiled pET32a plasmid in 1 % agarose gel. The DNA on the gel was stained with ethidium bromide. Binding reactions were performed in 50 mM MOPS-NaOH, pH7.4, containing 20 μM Fe²⁺or 100 μM H₂O₂ as indicated. Lanes: 1, Uuferritin only; 2, DNA only; 3, uuferritin plus DNA; 4, BSA plus DNA. The uuferritin-DNA complexes are indicated by the arrow. b The uuferritin protein protects DNA from hydroxyl radicals. Supercoiled pET32a plasmid was incubated with BSA (lanes 1 and 2) or with uuferritin (lanes 3 and 4) in 50 mM MOPS-NaOH, pH7.4, for 10 min at 25 °C. Hydroxyl radical generation via the Fenton reaction was achieved by adding 20 μM FeSO₄ and 100 μM H₂O₂ (lanes 2 and 4). Reactions were quenched and the uuferritin protein was degraded by Pronasek. Then DNA was analyzed on an agarose gel stained with ethidium bromide. (SC: supercoil DNA, N: nicked DNA). c The uuferritin suppressed the generation of the hydroxyl radical via the Fenton reaction. **p < 0.01 by U-test. Bars represent SD (n = 3)