| Autor: |
Souza LHC; Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil., Monteiro RGF; Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil., Guimarães WG; Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil., Gondim ACS; Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil., Sousa EHS; Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil., Diógenes ICN; Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60455-760 Fortaleza, CE, Brasil. |
| Jazyk: |
angličtina |
| Zdroj: |
Langmuir : the ACS journal of surfaces and colloids [Langmuir] 2024 Oct 01; Vol. 40 (39), pp. 20707-20714. Date of Electronic Publication: 2024 Sep 18. |
| DOI: |
10.1021/acs.langmuir.4c02792 |
| Abstrakt: |
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 2- ) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB 2- , on the surface, the mass of the attached TNB 2- 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 -2 for the TNB 2- ions (Γ TNB2- ). The ratio of this value to the surface coverage of CFL-1, Γ CFL-1 = 6.5 ± 0.6 pmol cm -2 (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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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