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

Several thioacylating reagents have been tested toward hydrolysis under con ditions suitable for protein modifications: 20-35 degreesC and buffered sol utions at pH 7.5-8.5, Aliphatic dithioesters are sufficiently stable in aqu eous media at room temperature (or below) if protein modification reaction time does not exceed 24 h, whereas at 35 degreesC reaction times must be li mited to a few hours. Kinetic data obtained in gelatin thioacylation at roo m temperature using aliphatic dithioesters and dithio acid are consistent w ith a second-order reaction rate with respect to amine concentration. The p H dependence of the second-order reaction rare constants indicate that dith ioester reacts exclusively with the free amine form of lysine residue, wher eas 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, t hus offering protein modification possibilities at pH 5-9. Thioacylation re action rates may be expressed as R = -(dA(t)/dt) = k[H3O+]-(b)A(t)(2)[thioa cylating agent] in which A, is the amine concentration at time t, constants k and b depending on the reagent nature.