The first term is the unfolding free energy when all ionizable residues are in their neutral form, the second term is the unfolding contribution when ionizable residues are charged to a reference pH value, pHref, and the third term is the unfolding contribution when changing the charge of the ionizable residues according to an actual pH value. In this particular case we use a pHref where all acids are uncharged and all bases are fully charged, which makes the contribution from the second term in the equation simple, and focus on the last term. The last term in the equation is the pH dependent term and can be calculated from the difference in charge between the folded and unfolded protein, ΔQ(pH) [26], as