Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
Paul Scherrer Institute, Forschungsstrasse 111, 5232 Villigen, Switzerland.
Molecules. 2022 May 15;27(10):3160. doi: 10.3390/molecules27103160.
The tight binding of Cu and Zn ions to superoxide dismutase 1 (SOD1) maintains the protein stability, associated with amyotrophic lateral sclerosis (ALS). Yet, the quantitative studies remain to be explored for the metal-binding affinity of wild-type SOD1 and its mutants. We have investigated the demetallation of Cu,Zn-SOD1 and its ALS-related G93A mutant in the presence of different standard metal ion chelators at varying temperatures by using an LC-ICP MS-based approach and fast size-exclusion chromatography. Our results showed that from the slow first-order kinetics both metal ions Zn and Cu were released simultaneously from the protein at elevated temperatures. The rate of the release depends on the concentration of chelating ligands but is almost independent of their metal-binding affinities. Similar studies with the G93A mutant of Cu,Zn-SOD1 revealed slightly faster metal-release. The demetallation of Cu,Zn-SOD1 comes always to completion, which hindered the calculation of the values. From the Arrhenius plots of the demetallation in the absence of chelators Δ = 173 kJ/mol for wt and 191 kJ/mol for G93A mutant Cu,Zn-SOD1 was estimated. Obtained high ΔH values are indicative of the occurrence of protein conformational changes before demetallation and we concluded that Cu,Zn-SOD1 complex is in native conditions kinetically inert. The fibrillization of both forms of SOD1 was similar.
铜和锌离子与超氧化物歧化酶 1（SOD1）紧密结合，维持着蛋白质的稳定性，与肌萎缩性侧索硬化症（ALS）有关。然而，对于野生型 SOD1 及其突变体与金属结合的亲和力，仍有待进行定量研究。我们采用基于 LC-ICP-MS 的方法和快速尺寸排阻色谱法，在不同温度下，用不同的标准金属离子螯合剂研究了 Cu、Zn-SOD1 及其与 ALS 相关的 G93A 突变体的去金属化作用。研究结果表明，从缓慢的一级动力学来看，两种金属离子 Zn 和 Cu 都在高温下同时从蛋白质中释放出来。释放的速度取决于螯合剂的浓度，但几乎与其金属结合亲和力无关。对 Cu、Zn-SOD1 的 G93A 突变体进行了类似的研究，结果表明金属释放速度稍快。Cu、Zn-SOD1 的去金属化作用总是会完全进行，这阻碍了 值的计算。在没有螯合剂的情况下，通过 Arrhenius 图对去金属化作用进行分析，得出 wt 型的 Δ = 173 kJ/mol，G93A 型的 Δ = 191 kJ/mol，Cu、Zn-SOD1 的突变体被估计为 191 kJ/mol。获得的高 ΔH 值表明在去金属化作用之前发生了蛋白质构象变化，我们得出结论，Cu、Zn-SOD1 复合物在动力学上处于天然状态下是惰性的。两种形式的 SOD1 的纤维化过程相似。
