title: 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.
authors.affiliation: Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay.
authors.forename: Florencia
authors.initials: F
authors.lastname: Sardi
authors.forename: Bruno
authors.initials: B
authors.lastname: Manta
authors.forename: Stephanie
authors.initials: S
authors.lastname: Portillo-Ledesma
authors.forename: Bernard
authors.initials: B
authors.lastname: Knoops
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Comini
authors.forename: Gerardo
authors.initials: G
authors.lastname: Ferrer-Sueta
mesh_terms: D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D005453:Fluorescence; D054477:Glutaredoxins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D054464:Peroxiredoxins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014346:Trypanosoma brucei brucei / Q000201:enzymology
chemical_list: D001643:Bridged Bicyclo Compounds; D054477:Glutaredoxins; D013438:Sulfhydryl Compounds; D054464:Peroxiredoxins; D003545:Cysteine; C028381:monobromobimane