Biological thermodynamic data for the calibration of differential scanning calorimeters: heat capacity data on the unfolding transition of ribonuclease a in solution

Abstract

Extensive measurements of the heat-capacity changes accompanying the unfolding transition of bovine pancreatic ribonuclease A in buffered aqueous solutions have been performed in a differential scanning calorimeter over a range of experimental conditions. The concentration of ribonuclease A varied from about 1 to 2 mass%. The pH varied from 1.8 to 5.0 at two glycine-HCl buffer concentrations: 0.1 and 0.2 M. Measurements were made on ribonuclease A obtained from various commercial sources. The heat capacity data were corrected for the thermal lag of the instrument and fitted by least-squares to a two-state model to determine the transition enthalpy and temperature, the heat-capacity change in the baseline, and the cooperativity of the transition. The transition temperature T m and enthalpy Δ H m determined from the fit of a two-state model to the transition profile increased linearly with pH from 311.9 ± 0.5 K and 308.2 ± 6.4 kJ mol −1 at pH = 2 to 335.4 ± 0.6 K and 408.9 ± 6.6 kJ mol −1 at pH = 4, where the uncertainties represent two standard deviations based on a linear leastsquares fit of Δ H m, and T m to pH. Values of T m and Δ H m were independent of the commercial source of ribonuclease A. The value of T m was independent of the buffer concentration but showed a slight dependence on the concentration of ribonuclease A. On the other hand, Δ H m was independent of concentration of ribonuclease A, but showed a slight dependence on the concentration of the glycine buffer solution. The heat capacity change obtained from the change in the transition baseline at T m was 3.4 ± 0.5 kJ mol −1 K −1 averaged over all determinations. The cooperativity of the transition, that is the ratio of the number of moles participating in the transition as determined from the two-state model to the actual number of moles in the sample, varied from 0.91 ± 0.02 at pH 2 to 1.07 ± 0.02 at pH 4 compared with unity for an ideal, two-state transition with a stoichiometry of one.