Determination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detection
| Autor: | Gerardo Ferrer-Sueta, Florencia Sardi, Stephanie Portillo-Ledesma, Bruno Manta, Marcelo A. Comini, Bernard Knoops |
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| Přispěvatelé: | Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República [Montevideo] (UCUR), Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Laboratorio de Química Teórica y Computacional Instituto de Química Biológica, Facultad de Ciencias, Laboratoire de Biologie Cellulaire, Université Catholique de Louvain = Catholic University of Louvain (UCL)-Institut des Sciences de la Vie, Center for Free Radical and Biomedical Research, Facultad de Ciensias, This work was supported by CSIC (Universidad de la República, Uruguay) to F.S. and G.F.-S. and by ANII (Agencia Nacional de Investigación e Innovación, Uruguay) |
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Nucleophilicity
[SDV]Life Sciences [q-bio] Inorganic chemistry Trypanosoma brucei brucei Biophysics 010402 general chemistry 01 natural sciences Biochemistry Fluorescence 03 medical and health sciences Bridged Bicyclo Compounds Reaction rate constant Nucleophile Humans pKa Reactivity (chemistry) Cysteine Sulfhydryl Compounds Molecular Biology Glutaredoxins 030304 developmental biology chemistry.chemical_classification 0303 health sciences Cell Biology Peroxiredoxins Hydrogen-Ion Concentration 0104 chemical sciences Catalytic cysteine chemistry Reagent Thiol Peroxiredoxin |
| Zdroj: | Analytical Biochemistry Analytical Biochemistry, Elsevier Masson, 2013, 435 (1), pp.74-82. ⟨10.1016/j.ab.2012.12.017⟩ |
| ISSN: | 0003-2697 1096-0309 |
| Popis: | International audience; A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-molecular-weight thiols. Nucleophilicity of the thiolate is measured as the pH-independent second-order rate constant of its reaction with mBBr. The ionization constants of the thiols are obtained through the pH dependence of either second-order rate constant or initial rate of reaction. For readily available thiols, the apparent second-order rate constant is measured at different pHs and then plotted and fitted to an appropriate pH function describing the observed number of ionization equilibria. For less available thiols, such as protein thiols, the initial rate of reaction is determined in a wide range of pHs and fitted to the appropriate pH function. The method presented here shows excellent sensitivity, allowing the use of nanomolar concentrations of reagents. The method is suitable for scaling and high-throughput screening. Example determinations of nucleophilicity and pK(a) are presented for captopril and cysteine as low-molecular-weight thiols and for human peroxiredoxin 5 and Trypanosoma brucei monothiol glutaredoxin 1 as protein thiols. |
| Databáze: | OpenAIRE |
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