Skip to Main content Skip to Navigation
New interface
Journal articles

Determination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detection.

Abstract : 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.
Document type :
Journal articles
Complete list of metadata

Cited literature [53 references]  Display  Hide  Download
Contributor : Mariella Botta Connect in order to contact the contributor
Submitted on : Monday, July 22, 2013 - 4:08:23 PM
Last modification on : Tuesday, January 18, 2022 - 2:50:07 PM
Long-term archiving on: : Wednesday, April 5, 2017 - 3:59:54 PM


 Restricted access
To satisfy the distribution rights of the publisher, the document is embargoed until : jamais

Please log in to resquest access to the document




Florencia Sardi, Bruno Manta, Stephanie Portillo-Ledesma, Bernard Knoops, Marcelo A Comini, et al.. Determination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detection.. Analytical Biochemistry, 2013, 435 (1), pp.74-82. ⟨10.1016/j.ab.2012.12.017⟩. ⟨pasteur-00847102⟩



Record views