from book 

Transition Metals

in Coordination

Environments

Although the protonation state of individual residues can be assessed experimentally

in solution with the help of NMR, [30] IR or in crystal with neutron diffraction,

[31] for computational methods, it is still a challenging task. This is due to the fact

that even small differences in free energy of protonation give rise to significant pKa

shifts (1.364 kcal/mol of energy at 298.15 K corresponds to a change of pKa by a

unit) [32].