Effect of ICL670A concentration on iron exchange between ICL670A-Fe and Fl-DFO.

Complexes of ICL670A-Fe were formed by mixing Fe:NTA (5 mM ferrous ammonium sulfate:35 mM nitrilotriacetate) with ICL670A in HBS to yield solutions containing 12 μM Fe and increasing concentrations of ICL670A (0, 12, 96, 192, 384 and 768 μM), followed by incubation for 1 hour at room temperature. At 0 minute, 20 μL of each chelator-Fe complex was mixed with 100 μL 2.5 μM Fl-DFO in HBS, and the fluorescence was monitored over time in a fluorescence plate reader. The final concentrations of both Fe and Fl-DFO were 2 μM in each system, whereas the concentration of ICL670A varied as shown from 0 through 2, 8, 16, 32, 64, to 128 μM.