Somack R, Nordeen S K, Eberhardt N L
Biochemistry. 1982 Oct 26;21(22):5651-60. doi: 10.1021/bi00265a041.
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.
为便于对甲状腺激素结合蛋白进行研究，我们合成并测试了光亲和类似物N-(乙基-2-重氮丙二酰基)-3,5,3'-三碘甲状腺原氨酸（EDM-T3）和N-(乙基-2-重氮丙二酰基)甲状腺素（EDM-T4）。L-EDM-T4和D-EDM-T4与人甲状腺素结合前白蛋白的结合亲和力分别为L-甲状腺素（L-T4）的4%和13.2%。作为比较，L-EDM-T3和D-EDM-T3对粗制大鼠肝核受体制剂的亲和力分别为L-三碘甲状腺原氨酸（L-T3）的0.1%和0.7%。通过十二烷基硫酸钠凝胶电泳判断，在254nm处对前白蛋白-[125I]-L-EDM-T4复合物进行光解导致[125I]-L-EDM-T4与前白蛋白发生共价连接。在无光解的情况下几乎未观察到标记。前白蛋白的光标记对高亲和力激素结合位点具有特异性，因为它（a）在过量3,5,3',5'-四碘甲状腺乙酸存在下光解时被完全阻断，（b）在高[125I]-L-EDM-T4浓度下饱和，（c）当激素结合位点先前已被前白蛋白的丹磺酰化阻断时被阻止，并且（d）被T3和T4竞争性阻断，抑制常数（K1）类似于这些配体的解离常数（Kd）。通过直接连接[125I]-L-EDM-T4或连接未标记的L-EDM-T4，然后用[125I]-L-T4滴定其余位点来分析前白蛋白光标记，结果表明在63-67%的位点占有率下光标记效率为54-61%。在用预辐照破坏重氮基团后，发现L-EDM-T4在进一步辐照后可标记前白蛋白；通过使用在87-91%位点占有率下的预辐照试剂可实现19-26%的光标记效率。这种不依赖卡宾的光附着对高亲和力激素结合位点也具有特异性。不依赖卡宾的过程机制可能涉及在甲状腺素环碘光诱导损失后通过自由基形成进行附着。因此，证明了L-T4与前白蛋白的特异性共价交联；然而，光标记效率远低于预辐照的EDM-T4。EDM-T4和T4均用于对未分级的人血清中的前白蛋白、甲状腺素结合球蛋白和白蛋白进行光标记。
