Evaluation of N-bromoacetyl-L-thyroxine as an affinity label for the thyroxine (T4)-binding site in human T4-binding...

The T4 analog N-bromoacetyl-L-T4 (BrAcT4) has been investigated as a possible affinity labeling reagent for identification of amino acids located within the T4-binding site in T4-binding globulin (TBG). As shown by fluorescence measurements involving displacement of 8-anilino-1-naphthalene-sulfonic acid from TBG, BrAcT4 is an effective competitor for the T4-binding site in TBG, with an association constant one seventh that of T4. Covalent modification of TBG by BrAcT4 was a slow process; after 48 h at a 10:1 molar ratio of [14C] BrAcT4 to TBG, incorporation of the 14C label reached 0.58 mol/mol protein or 77% of the theoretical value, correcting for 0.25 mol residually bound T4 in the original TBG sample. When [14C] BrAcT4 was reacted with TBG in the presence of T4, a partial inhibition of 25% in the degree of modification was obtained. The low inhibition of incorporation of label in the presence of T4 may be attributed to displacement of T4 from the binding site by BrAcT4 during the 20-h reaction time. To determine the effect of modification of the protein on binding activity, TBG was reacted with [14C]BrAcT4, and the binding capacity of modified TBG was determined by equilibrium dialysis. Three different TBG and three different [14C]BrAcT4 preparations were used. In two experiments, there was no reduction in binding capacity of modified TBG compared to that of control, although 0.6 and 0.48 mol label were incorporated per mol protein. In the third experiment, the decrease in binding capacity of modified TBG was 45% of the expected value. The lack of correspondence between the reduction in binding capacity and the degree of modification indicates that instead of reacting with amino acids within the T4-binding site, BrAcT4 derivatizes amino acids that are near but not actually part of the site. Covalent attachment of BrAcT4 to amino acids outside the T4-binding site places this compound in the category of an exoaffinity labeling reagent with regard to TBG and limits its usefulness for unequivocal identification of amino acids in the protein that participate directly in binding T4.