Fig. 2. Schematics of histidine chemistry concerning protonation, metal coordination and reaction with diethyl pyrocarbonate (DEPC). (I,II) The titration of a proton on the imidazole ring of His has a pK_a 6.5. The nitrogen shown losing the proton in the figure is the more likely of the two since it has slightly lower pK_a; however, depending on the local environment, either can be deprotonated. As shown in (III), it is possible for the deprotonated nitrogen to contribute a lone pair of electrons as a ligand in a metal coordination bond with a divalent transition metal ion such as Zn²⁺ or Cu²⁺. When both nitrogens are protonated (I), the ring is unable to participate as a ligand in metal bonding. Reacting unbound His residues with DEPC (IV) results in a carbethoxylation of the imidazole ring, rendering it unable to participate in coordination metal chemistry. Metal bound His will not react with DEPC. DEPC treatment is specific for His at pH 5.5 and at this pH, there will be few side chains bound to metal. EtOH, ethanol.