CONICET | Buscador de Institutos y Recursos Humanos

A method based on the reaction of thiols with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-molecular weight thiols. The method is based in the differential reactivity between thiol and thiolate and the concomitant difference in reaction rate [1]. Nucleophilicity of the thiolate is measured as thepH-independent second-order rate constant of its reaction with mBBr. The ionization constant of the thiols is obtained through the pH dependence of one of two possible kinetic parameters.For readily available thiols (low molecular weight thiols commercials), the apparent second- order rate constant is measured at different pHs and then plotted and fitted to an appropriate pH-function describing the observer number of ionization equilibriums. For protein thiols, theinitial rate of reaction is determined as a function of pH and fitted to the appropriate pH- function. The method presented herein shows excellent sensitivity allowing the use of nanomolar concentrations of reagents. In addition, this method is suitable for scaling it up for high-throughput screenings. Examples of determinations of nucleophilicity and pKa arepresented for low-molecular weight and proteins thiols. Finally, the strength of this analytical approach in the understanding of the chemical and structural features that determine protein thiol reactivity will be discussed using wild-type and mutants of E. coli thioredoxin as modelsystem.