Universidad de Buenos Aires - CONICET, Laboratorio de Biofísica Molecular. Instituto de Química y Fisicoquímica Biológicas, Buenos Aires, Argentina.
Universidad de Buenos Aires - CONICET, Laboratorio de Biofísica Molecular. Instituto de Química y Fisicoquímica Biológicas, Buenos Aires, Argentina.
Arch Biochem Biophys. 2023 Sep 1;745:109704. doi: 10.1016/j.abb.2023.109704. Epub 2023 Jul 30.
Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.
十二烷基硫酸钠（SDS）是一种众所周知的蛋白质变性剂。这种洗涤剂的一个不太为人知的性质是，它可以在亚变性浓度下激活或失活某些酶。在这项工作中，我们探索了 SDS 对超嗜热和嗜中温 Cu（I）ATPase 在磷脂和非变性洗涤剂混合胶束中重新构建的 ATPase 活性的影响。我们使用迭代程序来评估 SDS 在水相和胶束相之间的分配，从而可以确定从磷脂/洗涤剂混合物制备的胶束的组成。在存在不同量磷脂的情况下，用 SDS 孵育酶，结果表明，当磷脂的初始浓度增加时，需要更高的 SDS 浓度才能达到相同程度的失活。值得注意的是，我们发现，如果将其表示为胶束中 SDS 摩尔分数的函数，则在不同量的两亲物存在下获得的失活程度收敛到单个失活曲线。为了解释这个结果，我们提出了一个简单的模型，其中涉及处于平衡状态的活性和失活酶分子。该模型使我们能够估计失活过程的吉布斯自由能变化及其对胶束相中 SDS 摩尔分数的导数，后者是衡量酶对 SDS 的敏感性的指标。我们的结果表明，两种蛋白质的失活自由能变化相似。相反，超嗜热 ATPase 对 SDS 的敏感性显著降低，这表明嗜热性和对 SDS 的敏感性之间存在反比关系。
