The binding of spin-labeled propranolol and spin-labeled progesterone by orosomucoid

The binding of the spin-labeled propranolol and spin-labeled progesterone to human orosomucoid has been studied as a function of temperature by electron spin resonance (ESR) techniques. At 20 degrees C the association constants are 1.9 x 10(6) and 4.9 x 10(5) M-1, respectively. In each case, the binding is competitive with unlabeled ligand. Above about 50 degrees C the apparent association constant for both ligands decreases rapidly with increasing temperature. This is due to thermal denaturation of the orosomucoid, as was shown independently by ultraviolet absorption spectroscopy and differential scanning calorimetry. Below the denaturation region the number of binding sites per orosomucoid molecule remains constant at approx. 1. Examination of the thermodynamic parameters shows the progesterone binding at 37 degrees C to be essentially enthalpically driven, while the propranolol binding at 37 degrees C has a substantial entropic component.