Anserine buffering Anserine buffering (A) A schematic titration curve for anserine, with (B) an illustration of the species present in the reaction mixture during the titration. Anserine has three groups that can exist in both protonated and unprotonated forms: a carboxylic acid, an imidazole nitrogen and an amine. At the beginning of the titration, all the three groups are protonated and the predominant species in the reaction mix is (1). As base is added, the pH approaches the pKa for the first ionizable group, the carboxylic acid, and species (2) begins to form resulting in a plateau in the titration curve. Once the majority of molecules are in form (2), the pH will begin to rise again, before the second plateau occurs due to the formation of species (3). It is the equilibrium between species (2) and (3) that is relevant at biological pH. The final plateau occurs when the fully deprotonated species (4) is formed. Amanda L. Jonsson et al. Essays Biochem. 2017;61:401-427 ©2017 by Portland Press Ltd