Effect of a covalently attached synergistic anion on chelator-mediated iron-release from ovotransferrin: additional evidence for two concurrent pathways.

The mechanism by which the iron-transport protein transferrin releases its iron in vivo is presently unclear. In vitro studies have implicated two concurrent chelator-mediated iron-release pathways: one which is hyperbolic in nature, involving a conformational change in the protein as a rate limiting step, and a second which has been proposed to be first-order in nature and to involve initial release of a synergistic anion. We have examined the effect that an affinity-label analog of the synergistic anion has on chelator-mediated iron-release from this protein. A covalently attached anion would inhibit iron-release via any pathway in which anion release is a prerequisite to iron release. The present investigation examined the effect that the covalently attached anion had on iron-release to pyrophosphate (PPi) and N, N-bis(phosphonomethyl)glycine (DPG), two chelators which are believed to utilize both pathways concurrently. Results show that when the affinity-label anion is utilized, strictly hyperbolic data are obtained, with similar observed kmax values. This is strong support for the hypothesis of a common, chelator-independent rate-limiting step for the one available pathway. These results also support strongly the hypothesis that synergistic anion removal is a prerequisite step to iron-release via the second pathway.