Iron-binding proteins, exemplified by the transferrins, function to maintain the essential element iron in soluble, bioavailable and non-toxic form. Our goal is to identify the structural features and understand the molecular mechanisms underlying their biological activities. We will characterize, by thermodynamic, spectroscopic and physiologic studies, the metal-combining properties of single-sited, proteolytically-generated """"""""half-transferrins"""""""", in efforts to understand the consequences of transferrin's two-sited nature. We plan to continue probing the ligand structures of the protein's tightly-linked anion- and metal-binding sites by EPR, ENDOR and NMR spectroscopy. A hypothesis that lactoferrin, a relatively little studied member of the transferrin class of proteins, is involved in the generation of microbicidal hydroxyl radical will be examined by investigating its superoxide-dependent reactions using spin-trapping and related spectroscopic techniques. Because transferrin functions as an iron acceptor as well as an iron donor in its interaction with cells, we will ask whether receptors preferentially recognizing apotransferrin are required for iron delivery from cell to protein, in the way receptors for iron-bearing transferrin are needed for the transfer of iron from protein to cell. We intend to use monoclonal antibodies to attempt to discriminate between these two possible types of receptor, and to aid in their functional and molecular characterization. Purusing a new direction of research, we will investigate the iron-binding properties of uteroferrin, a protein once thought to be a natural """"""""half-transferrin"""""""" but now recognized as an iron-tyrosinate protein in its own right. Spectroscopic, thermodynamic, redox and anion-binding studies, designed to elucidate the characteristics of uteroferrin's metal center and the nature of its purple-to-pink transition, will be undertaken. Exchange of iron between uteroferrin and transferrin will also be examined, in keeping with our principal purpose of understanding the role of iron-binding proteins in the regulation of iron metabolism.
