Characterization of the unfolding dynamics of a recombinant type IA regulatory subunit (Riot) of cyclic adenosine monophosphate (CAMP)-dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RI alpha by CE in a buffer devoid of CAMP resulted in rapid dissociation of the complex from the original sample due to the high negative mobility of the ligand relative to receptor. This process enabled in-capillary generation of CAMP-stripped RI alpha, which was used to estimate the apparent dissociation constant (K-d) of 0.6 +/- 0.2 mu M. A comparison of RI alpha dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RI alpha-CAMP) and without (i.e., CAMP-stripped RI alpha) excess CAMP in the buffer during electromigration. The presence of CAMP in the buffer confirmed greater stabilization of the protein, as reflected by a higher standard free energy change (Delta G(U)degrees) of 10.1 +/- 0.5 kcal center dot mol(-1) and greater cooperativity in unfolding (m) of -2.30 +/- 0.11 kcal center dot mol(-1)M(-1). CE offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor-ligand complexes in free solution.