Abstract
Transferrins belong to an ancient family of extracellular proteins. The best-characterized transferrins are mammalian proteins that function in iron sequestration or iron transport; they accomplish these functions by having a high-affinity iron-binding site in each of their two homologous lobes. Insect hemolymph transferrins (Tsf1s) also function in iron sequestration and transport; however, sequence-based predictions of their iron-binding residues have suggested that most Tsf1s have a single, lower-affinity iron-binding site. To reconcile the apparent contradiction between the known physiological functions and predicted biochemical properties of Tsf1s, we purified and characterized the iron-binding properties of Drosophila melanogaster Tsf1 (DmTsf1), Manduca sexta Tsf1 (MsTsf1), and the amino-lobe of DmTsf1 (DmTsf1N). Using UV-Vis spectroscopy, we found that these proteins bind iron, but they exhibit shifts in their spectra compared to mammalian transferrins. Through equilibrium dialysis experiments, we determined that DmTsf1 and MsTsf1 bind only one ferric ion; their affinity for iron is high (log K' = 18), but less than that of the well-characterized mammalian transferrins (log K' ~ 20); and they release iron under moderately acidic conditions (pH50 = 5.5). Iron release analysis of DmTsf1N suggested that iron binding in the amino-lobe is stabilized by the carboxyl-lobe. These findings will be critical for elucidating the mechanisms of Tsf1 function in iron sequestration and transport in insects.
