Effect of thyroxine on molecular properties of native and dansylated thyroxine-binding globulin

The effect of thyroxine (T4) on the stability of thyroxine-binding globulin (TBG) was investigated by examining intrinsic (tryptophan) or extrinsic (DNS) fluorescence as structural probes to demonstrate the presence of an induced conformation change. A thermal transition was observed at 55 degrees C for TBG by Trp fluorescence, which is evidence for heat denaturation, whereas no transition was observed in the same temperature range for TBG-T4. Acidification of DNS-TBG at pH 4.4 produced a decrease in both Trp and DNS fluorescence whereas DNS-TBG-T4 at the same pH showed a large change in DNS fluorescence but no change in Trp fluorescence. Low guanidinium chloride concentration unfolded the unliganded molecule as indicated by a decrease of Trp and DNS fluorescence, whereas the fully occupied TBG showed only a decrease of DNS fluorescence. Exposure of TBG and TBG-T4 to trypsin showed a twofold increase in the rate of tryptic hydrolysis for TBG-T4 indicating that T4 binding either increased the exposure to the solvent of some lysyl or arginyl residues or increased their susceptibility to the trypsin action. These data provide additional evidence that T4 binding enhances the molecular stability of TBG. An effect on TBG conformation which was previously described probably explains the increased stability. The decrease in dansyl fluorescence of DNS-TBG-T4 probably is due to a local effect of acid or GdmCl unrelated to protein unfolding.