Ellman’s\nAssay on the Surface: Thiol Quantification\nof Human Cofilin‑1 Protein through Surface Plasmon Resonance

Oxidative stress on cysteine (Cys)-containing proteins\nhas been\nassociated with physiological disorders, as suggested for the human\ncofilin-1 (CFL-1) protein, in which the oxidized residues are likely\nimplicated in the aggregation process of α-synuclein, leading\nto severe neuronal injuries. Considering the relevance of the oxidation\nstate of cysteine, quantification of thiols may offer a guide for\nthe development of effective therapies. This work presents, for the\nvery first time, thiol quantification within CFL-1 in solution and\non the surface following classic and adapted versions of Ellman’s\nassay. The 1:1 stoichiometric Ellman’s reaction occurs between\n5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol\nof the cysteine residue, producing two 2-nitro-5-thiobenzoate (TNB<sup>2–</sup>) ions, one of which is released into the medium.\nWhile in solution, the thiol concentration was determined by the absorbance\nof the released TNB<sup>2–</sup>, on the surface, the mass\nof the attached TNB<sup>2–</sup> ion to the protein allowed\nthe quantification by means of the multiparametric surface plasmon\nresonance (MP-SPR) technique. The SPR angle change after the interaction\nof DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol\ncm<sup>–2</sup> for the TNB<sup>2–</sup> ions (Γ<sub>TNB2–</sub>). The ratio of this value to the surface coverage\nof CFL-1, Γ<sub>CFL‑1</sub> = 6.5 ± 0.6 pmol cm<sup>–2</sup> (also determined by MP-SPR), gave 4.1 as expected\nfor this protein, i.e., CFL-1 contains four Cys residues in its native\nform (reduced state). A control experiment with adsorbed oxidized\nprotein showed no SPR angle change, thus proving the reliability of\nadapting Ellman’s assay to the surface using the MP-SPR technique.\nThe results presented in this work provide evidence of the heterogenization\nof Ellman’s assay, offering a novel perspective for studying\nthiol-containing species within proteins. This may be particularly\nuseful to ensure further studies on drug-like molecules that can be\ncarried out with validated oxidized or reduced CFL-1 or other analogous\nsystems.