Thyroxine binding to human serum albumin immobilized on sepharose and effects of nonprotein albumin-binding plasma constituents

¹²⁵I-thyroxine (¹²⁵I-T₄) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied.¹²⁵I-T₄ binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19±0.53×10^–6 M and 2.69±0.78×10^–5 M, respectively. The number of binding sites of the high and the low affinity sites were 1.28±0.46 mol/mol and 23.5±9.7 mol/mol of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of¹²⁵I-T₄ to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-1-naphthalene sulfonic acid (ANS) inhibited the high affinity T₄ binding via reduction of the binding capacity. Unlabeled T₄ showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity T₄ binding to HSA-Sepharose.