Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil.
Langmuir. 2024 Oct 1;40(39):20707-20714. doi: 10.1021/acs.langmuir.4c02792. Epub 2024 Sep 18.
Oxidative stress on cysteine (Cys)-containing proteins has been associated with physiological disorders, as suggested for the human cofilin-1 (CFL-1) protein, in which the oxidized residues are likely implicated in the aggregation process of α-synuclein, leading to severe neuronal injuries. Considering the relevance of the oxidation state of cysteine, quantification of thiols may offer a guide for the development of effective therapies. This work presents, for the very first time, thiol quantification within CFL-1 in solution and on the surface following classic and adapted versions of Ellman's assay. The 1:1 stoichiometric Ellman's reaction occurs between 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the cysteine residue, producing two 2-nitro-5-thiobenzoate (TNB) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB, on the surface, the mass of the attached TNB ion to the protein allowed the quantification by means of the multiparametric surface plasmon resonance (MP-SPR) technique. The SPR angle change after the interaction of DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm for the TNB ions (Γ). The ratio of this value to the surface coverage of CFL-1, Γ = 6.5 ± 0.6 pmol cm (also determined by MP-SPR), gave 4.1 as expected for this protein, i.e., CFL-1 contains four Cys residues in its native form (reduced state). A control experiment with adsorbed oxidized protein showed no SPR angle change, thus proving the reliability of adapting Ellman's assay to the surface using the MP-SPR technique. The results presented in this work provide evidence of the heterogenization of Ellman's assay, offering a novel perspective for studying thiol-containing species within proteins. This may be particularly useful to ensure further studies on drug-like molecules that can be carried out with validated oxidized or reduced CFL-1 or other analogous systems.
半胱氨酸（Cys）含蛋白的氧化应激与生理紊乱有关，这一点在人类原丝切蛋白-1（CFL-1）蛋白中得到了证实，其中氧化残基可能与α-突触核蛋白的聚集过程有关，导致严重的神经元损伤。考虑到半胱氨酸氧化状态的相关性，硫醇的定量分析可能为开发有效的治疗方法提供指导。本工作首次在经典和改良的 Ellman 测定法中，在溶液中和表面上对 CFL-1 中的半胱氨酸进行了硫醇定量分析。5,5'-二硫代双（2-硝基苯甲酸）（DTNB）与半胱氨酸残基的游离巯基之间发生 1:1 化学计量的 Ellman 反应，生成两个 2-硝基-5-硫代苯甲酸（TNB）离子，其中一个释放到介质中。在溶液中，通过释放的 TNB 的吸光度来确定巯基浓度，而在表面上，与蛋白质结合的 TNB 离子的质量允许通过多参数表面等离子体共振（MP-SPR）技术进行定量。DTNB 与固定化 CFL-1 相互作用后，SPR 角度的变化给出了 TNB 离子（Γ）在表面上的 26.5 pmol cm 的覆盖度。将此值与 CFL-1 的表面覆盖度（Γ=6.5±0.6 pmol cm，也通过 MP-SPR 测定）相除，得到 4.1，这是该蛋白的预期值，即 CFL-1 在其天然形式（还原态）中含有四个半胱氨酸残基。用吸附氧化蛋白进行的对照实验没有显示 SPR 角度的变化，从而证明了使用 MP-SPR 技术将 Ellman 测定法适应表面的可靠性。本工作的结果提供了 Ellman 测定法异质性的证据，为研究蛋白质中含硫物种提供了新的视角。这对于确保进一步研究可以用验证的氧化或还原的 CFL-1 或其他类似系统进行的药物样分子可能特别有用。
