Time dependence of Fe transfer from L1 to apo-Tf.

Two solutions were prepared in HBS, one containing 12 μM Fe as Fe:NTA, and the other preformed L1-Fe complexes containing 100 μM L1 and 12 μM Fe. From each solution 20 μL was mixed with an equal volume of either HBS or HBS containing 25 μM apo-Tf, and incubated for 15 minutes at room temperature. To each mixture was added 100 μL 2.5 μM Fl-DFO in HBS and the fluorescence was monitored with time: ▪, Fe + apo-Tf; ●, L1-Fe + apo-Tf; ▾, L1-Fe; ▴, Fe alone. The final concentrations of L1, Fe, apo-Tf, and Fl-DFO in the reaction mixture were 14.3, 1.7, 3.6, and 1.8 μM, respectively.