Fig. 7. Ferritin protects DNA from iron-induced oxidative damage, in vitro. (A) Electrophoretic supercoil relaxation assay. The positions to which supercoiled (I), relaxed circular (II) and linear (III) monomeric DNAs migrated are indicated in the on the left. Reaction mixtures contained supercoiled pUC19 DNA in the following conditions: lane 1, DNA alone; lanes 2-7, DNA plus 50 µM FeCl₃ and 10 mM H₂O₂; lanes 3-7, recombinant H-ferritin at increasing concentrations (95 nM, 240 nM, 475 nM, 5 µM and 14.9 µM) in addition to the 50 µM FeCl₃ and 10 mM H₂O₂. (B) Graph of the mole fraction of form I DNA remaining at the end of the reaction as a function of the concentrations of ferritin and non-ferritin proteins. Reactions were performed as described in A, with the following ferritin samples: rH-ferritin (black square); apoferritin ferritin (black circle); recombinant 222 mutant ferritin (white square); and transferrin (black triangle). The CAP protein (white circle) was used in place of ferritin as a control for DNA-binding proteins. As controls for nonspecific protein effects, ferritin was replaced by ovalbumin (crossed white square), bovine serum albumin (white triangle) or chymotrypsin (black diamond) in duplicate reactions. All experiments were repeated three times. These data demonstrate that ferritin will protect DNA from iron-induced oxidative damage.