Binding of thyroid hormones to human hemoglobin and localization of the binding site.

Radiolabeled thyroid hormones were allowed to bind to erythrocyte cytosol and the complex was fractionated by Sephadex G-100 or by high-performance liquid chromatography (HPLC). On Sephadex G-100, four radioactive peaks (P1∼P4) were obtained, whereas HPLC gave only three radioactive peaks (P1∼P3). Chromatographic studies with human adult Hb and non-Hb cytosol protein fractions, which had been reacted with radiolabeled thyroid hormones, and immune precipitation with specific antisera for the hormones, confirmed that the first peak of Sephadex G-100 radioactivity was a mixture of Hb and non-Hb proteins, while the second peak was Hb. The third peak was free125I and the fourth peak was unbound125I-T3 or125I-T4. The third peak of HPLC was confirmed to be a mixture of free125I and unbound radiolabeled thyroid hormones. Scatchard analysis of the interaction between T4 and apo-Hb, and the α- and β-chains of human Hb suggested the presence of the specific binding site(s) for the hormone. Interaction between T4 and synthesized peptides, which constitute the heme pocket of the β-chain of Hb (β61–75, β71–85, β81–95), indicated that the T4 binding site of Hb resides within the heme-binding cavity. It is concluded that human erythrocyte cytosol does not contain “receptor” for thyroid hormones and cannot be a model for studying functions of cytosol “receptor” for the hormones; rather, it contains binding protein with large binding capacity, including Hb and non-Hb proteins, which possibly constitute a large reservoir for the hormone in blood.