Acidity and nucleophilicity in thiols by reaction with monobro- mobimane and fluorescence detection

FLORENCIA SARDI; BRUNO MANTA; STEPHANIE PORTILLO; JAVIER SANTOS; DIEGO SEBASTIAN VAZQUEZ; MARCELO COMINI; GERARDO FERRER-SUETA

Ciudad Autonoma de Buenos Aires

Congreso; XL Congreso Anual de la Sociedad Argentina de Biofísica; 2011

Fundación Instituto Leloir

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. Nucleophilicity of the thiolate is measured as the pH-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 weigth thiols commercials), the apparent second-order rate constant is measured at different pHs and then plotted and fitted to an appropiate pH-function describing the observer number of ionization equilibriums. For protein thiols, the initial 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 scalling it up for high-throughput screenings. Examples of determinations of nucleophilicity and pKa are presented for low-molecular weigth 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 model.