Protein thioacylation: 2. Reagent stability in aqueous media and thioacylation kinetics

Several thioacylating reagents have been tested toward hydrolysis under conditions suitable for protein modifications: 20-35 degreesC and buffered solutions at pH 7.5-8.5, Aliphatic dithioesters are sufficiently stable in aqueous media at room temperature (or below) if protein modification reaction time does not exceed 24 h, whereas at 35 degreesC reaction times must be limited to a few hours. Kinetic data obtained in gelatin thioacylation at room temperature using aliphatic dithioesters and dithio acid are consistent with a second-order reaction rate with respect to amine concentration. The pH dependence of the second-order reaction rare constants indicate that dithioester reacts exclusively with the free amine form of lysine residue, whereas dithiocarboxylate ion reacts with both amine and ammonium ion, probably through a more complex mechanism. Interestingly thioacylation using dithio acids may be obtained in pH near neutrality or in slightly acidic media, thus offering protein modification possibilities at pH 5-9. Thioacylation reaction rates may be expressed as R = -(dA(t)/dt) = k[H3O+]-(b)A(t)(2)[thioacylating agent] in which A, is the amine concentration at time t, constants k and b depending on the reagent nature.