Products Cited in Publications Worldwide Ellman's Assay on the Surface: Thiol Quantification of Human Cofilin-1 Protein through Surface Plasmon Resonance

Ellman's Assay on the Surface: Thiol Quantification of Human Cofilin-1 Protein through Surface Plasmon Resonance

Souza, Luiz HC; Monteiro, Rayssa GF; Guimarães, Wellinson G; Gondim, Ana CS; Sousa, Eduardo HS; Diógenes, Izaura CN

DOI: PMID:

Abstract

Oxidative stress on (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 , leading to severe neuronal injuries. Considering the relevance of the oxidation state of , 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’sreaction occurs between 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the residue, producing two 2-nitro-5-thiobenzoate (TNB2-) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB2-, on the surface, the mass of the attached TNB2- 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 with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm−2 for the TNB2- ions (ΓTNB2-). The ratio of this value to the surface coverage of CFL-1, ΓCFL-1 = 6.5 ± 0.6 pmol cm−2 (also determined by MP-SPR), gave 4.1 as expected for this protein|https://www.ambeed.com/protein.html, 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.

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