Lin T Y, Kim P S
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142.
Biochemistry. 1989 Jun 13;28(12):5282-7. doi: 10.1021/bi00438a054.
Thioredoxin contains a single disulfide bond that can be reduced without perturbing significantly the structure of the enzyme. Upon reduction of the disulfide, protein stability decreases. We have experimentally tested the expected linkage relationship between disulfide bond formation and protein stability for thioredoxin. In order to do this, it is necessary to measure the equilibrium constant for disulfide bond formation in both the folded and unfolded states of the protein. Using glutathione as a reference species, we have measured the equilibrium constant for forming the disulfide bond (effective concentration) in thioredoxin as a function of urea concentration. As a control, we show that urea per se does not interfere with our measurements of thiol-disulfide equilibrium constants. Comparison of the values obtained for disulfide bond formation in the folded and unfolded states with the free energies for unfolding oxidized and reduced thioredoxin using circular dichroism confirms the expected linkage relationship. The urea dependence of thiol-disulfide equilibria provides a sensitive assay for folded structure in peptides or proteins. The method should also be useful to evaluate the stabilizing or destabilizing effect of natural or genetically engineered disulfides in proteins. In future work, the effects of amino acid substitutions on disulfide bond formation could be evaluated individually in the native and unfolded states of a protein.
硫氧还蛋白含有一个二硫键，该二硫键可被还原而不会显著扰乱酶的结构。二硫键还原后，蛋白质稳定性降低。我们通过实验测试了硫氧还蛋白中二硫键形成与蛋白质稳定性之间预期的联系关系。为了做到这一点，有必要测量蛋白质折叠态和未折叠态中二硫键形成的平衡常数。以谷胱甘肽作为参考物质，我们测量了硫氧还蛋白中形成二硫键（有效浓度）的平衡常数作为尿素浓度的函数。作为对照，我们表明尿素本身不会干扰我们对硫醇 - 二硫键平衡常数的测量。使用圆二色性将折叠态和未折叠态中二硫键形成所获得的值与氧化型和还原型硫氧还蛋白展开的自由能进行比较，证实了预期的联系关系。硫醇 - 二硫键平衡对尿素的依赖性为肽或蛋白质中的折叠结构提供了一种灵敏的检测方法。该方法对于评估蛋白质中天然或基因工程二硫键的稳定或去稳定作用也应该是有用的。在未来的工作中，可以在蛋白质的天然态和未折叠态中分别评估氨基酸取代对二硫键形成的影响。
