Given the above interpretation of our results, the protonation event already takes prior to the complexation process. The benzoic acid becomes protonated when being exposed to the protein crystallisation buffer medium. In contrast, the benzoic acid appears in the unprotonated form when being present in the ITC buffer. Under these conditions, the benzoic acid cannot bind to the ATP pocket of PKA (at least in the protonated form) which makes the missing signal in the ITC experiments comprehensible. The determination of pH value of the crystallisation buffer further strengthens our hypothesis.