甲状腺素与蛋白质的相互作用。甲状腺素和三碘甲状腺原氨酸与人甲状腺素结合球蛋白的相互作用。

Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin.

作者信息

Korcek L, Tabachnick M

出版信息

J Biol Chem. 1976 Jun 25;251(12):3558-62.

Abstract

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.

摘要

通过平衡透析研究了温度对甲状腺素和三碘甲状腺原氨酸与甲状腺素结合球蛋白结合的影响。在透析混合物中加入卵清蛋白可稳定甲状腺素结合球蛋白，防止其在平衡透析过程中出现结合活性损失。卵清蛋白自身对甲状腺激素的结合非常弱，在分析实验结果时其结合可忽略不计。在pH 7.4和37℃的0.06 M磷酸钾/0.7 mM EDTA缓冲液中，甲状腺素在单一结合位点与甲状腺素结合球蛋白结合，其表观缔合常数为：5℃时，K = 4.73±0.38×10¹⁰ M⁻¹；25℃时，K = 1.55±0.17×10¹⁰ M⁻¹；37℃时，K = 9.08±0.62×10⁹ M⁻¹。三碘甲状腺原氨酸结合数据的Scatchard图表明，该化合物与甲状腺素结合球蛋白的结合比甲状腺素更为复杂。三碘甲状腺原氨酸结合数据可通过假设存在两类不同的结合位点来拟合。在5℃和pH 7.4时，对数据进行非线性回归分析得到的值为n1 = 1.04±0.10，K1 = 3.35±0.63×10⁹ M⁻¹，n2 = 1.40±0.08，K2 = 0.69±0.20×10⁸ M⁻¹。在25℃时，结合常数的值为n1 = 1.04±0.38，K1 = 6.5±2.8×10⁸ M⁻¹，n2 = 0.77±0.22，K2 = 0.43±0.62×10⁸ M⁻¹。在37℃时，观察到的曲率较小，估计的结合常数为n1 = 1.02±0.06，K1 = 4.32±0.59×10⁸ M⁻¹，n2K2 = 0.056±0.012×10⁸ M⁻¹。当n1固定为1时，在25℃时得到的其他三个结合常数的值为K1 = 6.12±0.35×10⁸ M⁻¹，n2 = 0.72±0.18，K2 = 0.73±0.36×10⁸ M⁻¹；在37℃时，K1 = 3.80±0.22×10⁸ M⁻¹，n2 = 0.44±0.22，K2 = 0.43±0.38×10⁸ M⁻¹。在37℃和pH 7.4时，甲状腺素与甲状腺素结合球蛋白结合的热力学值为ΔG⁰ = -14.1 kcal/mol，ΔH⁰ = -8.9６ kcal/mol，ΔS⁰ = +16.7 cal·℃⁻¹·mol⁻¹。对于37℃的三碘甲状腺原氨酸，其在主要结合位点结合的热力学值为ΔG⁰ = -1２.3 kcal/mol，ΔH⁰ = -11.9 kcal/mol，ΔS⁰ = +1.4 cal·℃⁻¹·mol⁻¹。结合的pH依赖性测量表明，甲状腺素和三碘甲状腺原氨酸在生理pH范围（pH 6.8至7.7）内结合程度最大。