In vitro formation of apparent covalent complexes between L-triiodothyronine and plasma protein

Nondissociable complexes between¹²⁵I-labeled T₃ (¹²⁵I-T₃) and plasma proteins were observed after incubation of¹²⁵I-T₃ with plasma diluted 200-fold with phosphate buffer. The formation of these complexes was enhanced in the presence of Cu²⁺ and flavin mononucleotide and inhibited by reducing agents such as propylthiouracil, thiosulfate, dithiothreitol, and glutathione. The¹²⁵I-T₃ protein complexes could be disrupted after digestion with pronase. T₃ itself was identified as a constituent of the pronase digests by co-chromatography with authentic¹³¹I-T₃ to a constant isotopic ratio across the T₃ area. Since treatment with 0.2% sodium dodecylsulfate or 8M urea did not disrupt the complexes, the T₃-protein bond appears to have some of the characteristics of a covalent bond between hormone and protein.