Cytochrome c Dimer Dissociation Kinetics

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 (f_Dimer) and monomer (f_Monomer) were evaluated from their relative peak heights at 280 nm in the FPLC chromatogram. Plots of f_Dimer and f_Monomer versus time were fit to a single exponential to determine the rate constant for conversion of dimer to monomer (k_DM). Eyring plots of k_DM were then fit to eq 1 using a reference temperature (T₀) of 310.15 K.
In eq. 1k_B and h are the Boltzmann and Planck constants, respectively. ${∆H}_{T_0}^{‡}$ and ${∆S}_{T_0}^{‡}$ are the difference in enthalpy and entropy between the ground state and the transition state. ${∆C}_p^{‡}$ is the difference in heat capacity between the ground state and the transition state (TS) at constant pressure.