Fasudil was used as high-affinity PKA inhibitor and displacement ligand. As a control for both the displacement titration and to verify the activity of the protein PKA, PKA was titrated with fasudil alone. Although no thermodynamic parameters have been published for human PKA, they have been published for the homologous variant from Cricetulus griseus, which differs only at six terminal amino acids. The result of this control measurement revealed a stoichiometry of N = 0.9, meaning that most of the protein is intact and active (Figure , left column). The overall ΔG of -37.3 kJ/mol is also in close proximity of the hamster variant (ΔG = -36.1 kJ/mol). In order to test the benzoic acid fragment, we firstly performed a direct titration of PKA and benzoic acid (Figure , middle column). The used concentrations were 140 µM for the protein and 2.8 mM for the benzoic acid, respectively. Unfortunately, a binding signal could not be detected.