.I. Med Chem. 1986, 29, 648454
648
the crystal y axis). The list of contacts are given in Tables I and 11. Table I11 lists meta contacts for an iodinated biphenyl system (numbered as in Chart I) calculated on the basis of the coordinates for the 2,2',4, i',6,6'-1fCB. Binding Assays. The thyroxine reference standard and all analogues were dissolved in methanol in the concentration range from 0.1 to 10 KM and were stored at 4 "C. The stock solution of TBPA contained 0.127 mg/mL of the protein in imidazole acetate buffer. The stock solution was further diluted to 200 nM prior to use. No change of the binding capacity of the TBPA stock solution due to storage was observed. However, the 200 nM solution quickly lost binding capacity and had to be prepared immediately prior to use. T o characterize the affinity of L-T,for the high affinity binding site of TBPA, Scatchard analysis was carried out. Tn contrast to the procedure of the competitive binding assay, the amount of ['251]-~-T4 was varied together with the increasiiig concentration of unlabeled T4 (1.9-940 nM) to achieve a constant specific activity (320 pCi/nmol). The TBPA Concentration was 10 nM. Incubation was carried out a t 25 "C. Noiispecific binding was measured by adding excess (940 nM) unlabeled I -7'& A binding assay using the gel filtration procedure described by Somack et al.', was used to measure the ability of various halogenated biphenyls and other analogues to displace [ 1251]-~-'r4 from the high-affinity TBPA binding site. The assay mixture contained imidazole acetate buffer, 10 nM TBPA, 0.57 nM ['2'1]-~~-'14 with a total activity of
