The pH dependence of the thermodynamics of the interaction of diazepam with human serum albumin.

The heat effect associated with the binding of diazepam to human serum albumin has been measured from pH 5.5 to 9.5 using flow microcalorimetry. The number of protons released upon the binding of one molecule of diazepam to one molecule of albumin was determined from acid-base titration experiments. In addition, the heat associated with the combination of protons with serum albumin was measured calorimetrically. The results of the experiments using these two techniques were used to correct the measured heat of binding and to obtain what will be called here delta H0obs values. These delta H0obs values are strongly pH-dependent: up to pH 7.5 a value of -24.3 (+/- 0.5) kJ X mol-1 is found, whereas at pH 9.5 the value is -44.3 (+/- 1.2). This pH dependence is different from the pH dependence of the induced CD signal of the albumin-diazepam complex. A model is presented in which the pH dependence of delta H0obs is explained. This model takes into account the N-B (neutral to base) conformational change in albumin around neutral pH. Parameters in this model are KN and KB (representing the affinity of diazepam for albumin in the N and B conformation, respectively), delta H0N and delta H0B which are the corresponding standard enthalpy changes, f which represents the fraction of protein in the B conformation, and delta H0(N-B) which represents the heat associated with the N-B transition.