Chemical, but not functional, differences between the iron-binding sites of rabbit transferrin

Abstract

The interaction between iron and rabbit transferrin was investigated by three methods, (1) by studying iron release mediated by phosphatic compounds and iron chelators, (2) by determining the distribution of iron between the binding sites in the presence of chelators and (3) by measuring iron uptake from two iron-binding sites by rabbit reticulocytes. The distribution of iron between diferric transferrin and the two forms of monoferric transferrin was determined by electrophoresis in polyacrylamide gel containing 6 M urea. Iron release mediated by ATP, ADP, 2,3-diphosphoglycerate or citrate occurred at a single rate in a manner indicating that iron was released at the same rate from the two iron-binding sites and that iron release from either site was independent of that from the other. By contrast, iron release mediated by nitrilotriacetate, pyrophosphate or oxalate occurred at different rates from the two binding sites, more rapid release occurring from one site with nitrilotriacetate and pyrophosphate and from the other site with oxalate. The rates of iron release mediated by the above substances varied with incubation temperature and pH. In the presence of nitrilotriacetate, pyrophosphate or oxalate iron bound preferentially to that site from which it was more slowly released by the chelator. When transferrin was incubated with reticulocytes iron was taken up much more rapidly with diferric transferrin than from monoferric transferrin, but there was no evidence of preferential uptake of iron from either binding site. It is concluded that there are distinct chemical differences between the two iron-binding sites of rabbit transferrin. However, they were not reflected by functional differences under the conditions of incubation with reticulocytes used in this work. The results for iron release from transferrin mediated by the action of phosphatic compounds and chelators support the concept that protonation is involved in the rate-limiting step in the iron-release process.