Grimaldi S, Robbins J
Endocrinol Exp. 1982 Nov;16(3-4):273-81.
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.
通过检测作为结构探针的内源（色氨酸）或外源（丹磺酰）荧光来研究甲状腺素（T4）对甲状腺素结合球蛋白（TBG）稳定性的影响，以证明诱导构象变化的存在。通过色氨酸荧光观察到TBG在55℃有一个热转变，这是热变性的证据，而在相同温度范围内，TBG-T4未观察到转变。在pH 4.4对DNS-TBG进行酸化会导致色氨酸和丹磺酰荧光均降低，而相同pH下的DNS-TBG-T4丹磺酰荧光有很大变化，但色氨酸荧光无变化。低浓度胍盐酸盐会使未结合配体的分子展开，表现为色氨酸和丹磺酰荧光降低，而完全结合的TBG仅表现为丹磺酰荧光降低。将TBG和TBG-T4暴露于胰蛋白酶表明，TBG-T4的胰蛋白酶水解速率增加了两倍，这表明T4结合要么增加了一些赖氨酰或精氨酰残基与溶剂的接触，要么增加了它们对胰蛋白酶作用的敏感性。这些数据提供了额外的证据，证明T4结合增强了TBG的分子稳定性。先前描述的对TBG构象的影响可能解释了稳定性的增加。DNS-TBG-T4的丹磺酰荧光降低可能是由于酸或盐酸胍的局部作用，与蛋白质展开无关。
