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240919s2024 xx |||||o 00| ||eng c |
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|a 10.1021/acs.langmuir.4c02792
|2 doi
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|a pubmed24n1558.xml
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|a (DE-627)NLM377798088
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|a (NLM)39292813
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|a DE-627
|b ger
|c DE-627
|e rakwb
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|a eng
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| 100 |
1 |
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|a Souza, Luiz H C
|e verfasserin
|4 aut
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| 245 |
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|a Ellman's Assay on the Surface
|b Thiol Quantification of Human Cofilin-1 Protein through Surface Plasmon Resonance
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|c 2024
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|a Text
|b txt
|2 rdacontent
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|a ƒaComputermedien
|b c
|2 rdamedia
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|a ƒa Online-Ressource
|b cr
|2 rdacarrier
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|a Date Completed 01.10.2024
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|a Date Revised 05.10.2024
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|a published: Print-Electronic
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|a Citation Status MEDLINE
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|a 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 (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 DTNB 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, 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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|a Journal Article
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|a Sulfhydryl Compounds
|2 NLM
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|a Cofilin 1
|2 NLM
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|a Dithionitrobenzoic Acid
|2 NLM
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| 650 |
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7 |
|a 9BZQ3U62JX
|2 NLM
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| 650 |
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|a CFL1 protein, human
|2 NLM
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| 650 |
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|a Cysteine
|2 NLM
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| 650 |
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|a K848JZ4886
|2 NLM
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| 700 |
1 |
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|a Monteiro, Rayssa G F
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Guimarães, Wellinson G
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Gondim, Ana C S
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Sousa, Eduardo H S
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Diógenes, Izaura C N
|e verfasserin
|4 aut
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| 773 |
0 |
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|i Enthalten in
|t Langmuir : the ACS journal of surfaces and colloids
|d 1999
|g 40(2024), 39 vom: 01. Okt., Seite 20707-20714
|w (DE-627)NLM098181009
|x 1520-5827
|7 nnns
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| 773 |
1 |
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|g volume:40
|g year:2024
|g number:39
|g day:01
|g month:10
|g pages:20707-20714
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| 856 |
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|u http://dx.doi.org/10.1021/acs.langmuir.4c02792
|3 Volltext
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|d 40
|j 2024
|e 39
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|c 10
|h 20707-20714
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