Biological thermodynamic data for the calibration of differential scanning calorimeters: heat capacity data on the unfolding transition of β-lactoglobulin in solution

Abstract

Differential scanning calorimetry (DSC) measurements of the unfolding transition of β-lactoglobulin in HCl-glycine-buffered solutions have been performed over a temperature range from 353.0 K at pH 2.3 to 362.2 K at pH 3.5. Three values for the van't Hoff transition enthalpies were calculated as follows: (1) from the fit of a two-state transition model to the heat capacity measurements (Δ H vf); (2) from the van't Hoff plot of ln(1/ K) 1/ T where K is the transition equilibrium constant; and (3) from the equation Δ H ve = 4.00 RT 2 m C max p/ A p where R = 8.31451 J mol −1 K −1, T m is the transition temperature, C max p is the peak maximum, and A p is the peak area. The best linear fit of the van't Hoff enthalpies to the transition temperatures, T m, was obtained with Δ H vf and is Δ H vf (kJ mol −1) = (438.6 ±2.8) +(1.8±1.0)( T m −358.0). Calorimetric transition enthalpies were determined from the transition peak area using an extrapolated sigmoidal baseline and using an extrapolated straight baseline. The best linear dependence of the calorimetric enthalpy on T m was obtained with the sigmoidal baseline (Δ H s) and is Δ H s(kJ mol −1) = (427.1 ± 4.2) + (0.5 ±1.5)( T m-358.0). Linear least-squares fits of Δ H vf and Δ H s to T m were independent of the DSC scan rate, the source of β-lactoglobulin, the buffer concentration from 0.1 to 0.2 M, and the concentration of the protein from 0.03 to 0.45 mM. The transition temperature exhibits a linear dependence on pH and a slight dependence on concentration. Cooperativity of the transition is 0.974 ± 0.007 while the average heat capacity change of the solution accompanying the transition is 13.5±7.8 kJ mol −1 K −1 over this temperature range.