Thermal denaturation of ribonuclease A (RNase A) complex with cytidine 3'-monophosphate (3'CNIP) was studied by differential scanning calorimetry (DSC). The kinetic and binding studies of RNase A with cytidine 2',3'-cyclic phosphate (cCMP) as a substrate, and 3'CMP as a ligand were also investigated by difference spectrophotometry. The obtained kinetic saturation curve reveals the occurrence of an anomalous non-hyperbolic shape at high substrate concentrations, and a biphasic binding isotherm. These phenomena indicate that a conformational change is occurring with RNase A during the hydrolysis of cCMP. A combination of kinetic and thermodynamic studies tends to elucidate the reasons for the formation of a non-hyperbolic behavior in a kinetic saturation curve. The thermal profile of the enzyme-3'CMP complexes shows a splitting of two distinct peaks with different structural stabilities of melting points (T-m) of 325 and 337 K. The bifurcate appearance of DSC profile of RNase A-3'CMP complexes manifests a physical view of a light kinetic structural transition. It is worthy to note, the direct binding (not via enzymatic reaction) of enzyme with 3'CMP indicates single DSC profile and monophasic binding isotherm. (C) 2003 Elsevier B.V. All rights reserved.