THE ANION REQUIREMENT FOR IRON RELEASE FROM TRANSFERRIN IS PRESERVED IN THE RECEPTOR TRANSFERRIN COMPLEX

Rates of iron release from both sites of free transferrin at pH 7.4 ar e critically dependent upon ionic strength, because release appears to require binding of a simple nonchelating anion such as chloride to a kinetically active site of the protein. This site is distinct from the synergistic anion-binding site, occupancy of which is required for bi nding of iron to occur at all. Complexing of transferrin to its recept or also modulates release of iron, but in a more complex fashion. At e xtracellular pH, 7.4, receptor retards release, but at the pH of the e ndosome in which release occurs within the cell, 5.6, receptor acceler ates release. The present study was undertaken to determine whether th e kinetically active anion requirement is maintained at pH 5.6 and whe ther the effects of anion binding and receptor binding are independent of each other. A spectrofluorometric method was developed to monitor release of iron from C-terminal monoferric human transferrin and its c omplex with the transferrin receptor. At pH 5.6, as at pH 7.4, profile s of iron release to pyrophosphate from free and from receptor-complex ed monoferric transferrin show curvilinear dependence on pyrophosphate concentration, consistent with a previously described kinetic scheme and suggestive of a similar release mechanism in all cases. Furthermor e, at pH 5.6 release rates depend upon anion (chloride) concentration in free and in receptor-complexed transferrin as in free transferrin a t pH 7.4, extrapolating nearly to zero as chloride concentration appro aches zero. The enhancing effect of receptor on release is displayed a t all concentrations of chloride tested, indicating that the release-p romoting effects of receptor and chloride are independent of each othe r. Since release is thought to occur from a lobe of transferrin when t he two domains enclosing the binding site of the lobe rotate about the ir hinging strands to an ''open'' conformation, one possibility is tha t in the C-terminal lobe the anion- or receptor-binding sites, or both , are located in the hinging strands.