LIGAND-INDUCED BIPHASIC THERMAL-DENATURATION OF RNASE-A

DSC measurements have been accomplished in aqueous solutions of bovine pancreatic ribonuclease A (RNAase A) in the presence of subsaturating amounts of 3' cytidine monophosphate (3' CMP) and 2' cytidine monopho sphate (2' CMP) at pH 5.0 and 5.5. In these conditions the experimenta l profiles do not conform to a one-step unfolding process. It can be e mphasized, as a general phenomenon, that a strong linkage between the temperature-induced protein unfolding and the ligand binding, when the ligand is less than the saturation level, causes marked distortions f rom a two-state transition. A purely equilibrium thermodynamic analysi s gives a correct account of this behaviour and allows to simulate cal orimetric curves. It is thus possible to obtain, in an indirect manner , information about the thermodynamic parameters concerning the bindin g process, namely the association constant and the binding enthalpy. T he values of K(b) and DELTA(b)H for 3' CMP and 2' CMP, so determined, are consistent with the literature data.