Human cytc DSD dissociation kinetics were studied using previous published methods.27 (link) Briefly, following isolation of pure dimer using the BioRad Enrich SEC70 column at 4 °C in 50 mM potassium phosphate (pH 7), solutions of dimer were incubated at 40, 45, 50, 55 and 60 °C in a Julabo F12-ED refrigerated/heating circulator. Dissociation from dimer to monomer was measured at each time point by size-exclusion chromatography of 100–150 μL aliquots using the SEC70 column. The fractions of dimer (fDimer) and monomer (fMonomer) were evaluated from their relative peak heights at 280 nm in the FPLC chromatogram. Plots of fDimer and fMonomer versus time were fit to a single exponential to determine the rate constant for conversion of dimer to monomer (kDM). Eyring plots of kDM were then fit to eq 1 using a reference temperature (T0) of 310.15 K.
In eq. 1kB and h are the Boltzmann and Planck constants, respectively. HT0 and ST0 are the difference in enthalpy and entropy between the ground state and the transition state. Cp is the difference in heat capacity between the ground state and the transition state (TS) at constant pressure.