Figure 2. ITC of wild-type ferrochelatase with mesoporphyrin.

(A) Titration in Bicine buffer. Experiments were performed at 25 °C. The plot represents the integral heat for the titration in Bicine buffer of mesoporphyrin (336 μM, 8.6 μl/injection) into ferrochelatase (15 μM) versus the mesoporphyrin to ferrochelatase molar ratios (each point represents an injection). The continuous line is a guide for the eye and the broken lines show the crossing at a 1:1 binding stoichiometry. (B) Effect of buffer on the observed enthalpies. The observed enthalpy changes for the binding reaction of mesoporphirin to ferrochelatase (ΔH_obs) in the three studied buffers are plotted as a function of the ionization enthalpy of the respective buffer as reported in [44]. The slope of the fitted curve yields the number of protons released by the buffer and the intercept yields the binding enthalpy (ΔH_o) that would be observed in a buffer with ΔH_ionization=0 [see eqn (1) in the text].