Thyroxine-binding globulin: characterization of the binding site with a fluorescent dye as a probe

The fluorescent dye 1,8-anilinonaphthalenesulfonate competed with thyroxine for binding to thyroxine-binding globulin. Fluorescence analysis indicated that the dye bound to the globulin in a molar ratio of 1:1 and with an association constant (at 23 degrees C) of 4.19 x10(6)M(-1), and that thyroxine bound to the globulin in a molar ratio of 1:1 and with an association constant (at 23 degrees C) of 2.35x10(10)M(-1). Displacement of globulin-bound dye by thyroxine was shown by fluorescence quenching, and displacement of globulin-bound thyroxine by dye was demonstrated by ultrafiltration.