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

Abstract

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 free 125I and the fourth peak was unbound 125I-T3 or 125I-T4. The third peak of HPLC was confirmed to be a mixture of free 125I and unbound radiolabeled thyroid hormones. Scatchard analysis of the interaction between T4 and apo-Hb, and the alpha- and beta-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 beta-chain of Hb (beta 61-75, beta 71-85, beta 81-95), 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.