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 degrees C 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 degrees C 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 rate 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 = -(dAt/dt) = k[H3O+](-b)At2[thioacylating agent] in which At is the amine concentration at time t, constants k and b depending on the reagent nature.
Levesque
G
G
Centre de Recherche, Université de Bretagne-Sud, Boulevard Flandres-Dunkerque, F-56325 Lorient, France.
Arsène
P
P
Fanneau-Bellenger
V
V
Pham
T N
TN
D000215:Acylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D014867:Water
D007202:Indicators and Reagents; D011506:Proteins; D013438:Sulfhydryl Compounds; D014867:Water; D008239:Lysine