Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles

Abstract

A⁵⁹Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with⁵⁹Fe,¹²⁵I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for⁵⁹Fe-citrate of 3.63×10⁹ m ⁻¹ and with 6.6×10⁻¹² moles of iron bound per mg protein and the other with a constant of 3.96×10⁸ m ⁻¹ and 1.0×10⁻¹² moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the⁵⁹Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with⁵⁶Fe and an equivalent amount of⁵⁹Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.