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

1988; Elsevier BV; Volume: 128; Linguagem: Inglês

10.1016/0040-6031(88)85371-1

1872-762X

Frederick P. Schwarz, William H. Kirchhoff,

Protein Structure and Dynamics

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 Tm and enthalpy ΔHm 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 ΔHm, and Tm to pH. Values of Tm and ΔHm were independent of the commercial source of ribonuclease A. The value of Tm was independent of the buffer concentration but showed a slight dependence on the concentration of ribonuclease A. On the other hand, ΔHm 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 Tm 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.