Photoaffinity labeling of human thyroxine-binding prealbumin with thyroxine and N-(ethyl-2-diazomalonyl)thyroxine.
To facilitate studies of thyroid hormone-binding proteins, we have synthesized and tested the photoaffinity analogues N-(ethyl-2-diazomalonyl)-3,5,3'-triiodothyronine (EDM-T3) and N-(ethyl-2-diazomalonyl)thyroxine (EDM-T4). The binding affinities of L-EDM-T4 and D-EDM-T4 to human thyroxine-binding prealbumin were 4% and 13.2%, respectively, that of L-thyroxine (L-T4). For comparison the affinities of L-EDM-T3 and D-EDM-T3 to crude rate liver nuclear receptor preparation were 0.1% and 0.7%, respectively, that of L-triiodothyronine (L-T3). Photolysis of prealbumin-[125I]-L-EDM-T4 complexes at 254 nm resulted in covalent linkage of [125I]-L-EDM-T4 to prealbumin as judged by sodium dodecyl sulfate gel electrophoresis. Virtually no labeling was observed in the absence of photolysis. Photolabeling of prealbumin was specific for the high-affinity hormone binding site since it was (a) completely blocked during photolysis in the presence of excess 3,5,3',5'-tetraiodothyroacetic acid, (b) saturated at high [125I]-L-EDM-T4 concentrations, (c) prevented when the hormone binding site had been previously blocked by dansylation of prealbumin, and (d) blocked competitively by T3 and T4 with inhibition constants (K1) similar to the dissociation constants (Kd) for these ligands. Analysis of prealbumin photolabeling by direct attachment of [125I]-L-EDM-T4 or attachment of unlabeled L-EDM-T4 followed by titration of the remaining sites with [125I]-L-T4 indicated a photolabeling efficiency of 54-61% at 63-67% site occupancy. After destruction of the diazo group by preirradiation, L-EDM-T4 was found to label prealbumin after further irradiation; 19-26% photolabeling efficiency could be achieved by using preirradiated reagent at 87-91% site occupancy. This carbene-independent photoattachment was also specific for a high-affinity hormone binding site. The mechanism of the carbene-independent process may involve attachment via radical formation following photoinduced loss of the thyronine ring iodine. Accordingly, specific covalent cross-linking of L-T4 to prealbumin was demonstrated; however, the photolabeling efficiency was much lower than that with preirradiated EDM-T4. Both EDM-T4 and T4 were employed to photolabel prealbumin, thyroxine binding globulin, and albumin in unfractionated human serum.
D000345:Affinity Labels; D001391:Azo Compounds / Q000378:metabolism*; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003979:Diazonium Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011956:Receptors, Cell Surface / Q000378:metabolism; D011988:Receptors, Thyroid Hormone; D013974:Thyroxine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013975:Thyroxine-Binding Proteins / Q000378:metabolism*
D000345:Affinity Labels; D001391:Azo Compounds; D001798:Blood Proteins; D003979:Diazonium Compounds; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D013975:Thyroxine-Binding Proteins; C036862:N-(ethyl-2-diazomalonyl)thyroxine; D013974:Thyroxine