Note that ∆∆G p,s (A H , A -) implicitly contains two terms. The first (∆∆G env ) represents the free energy of dissociating a proton within a protein relative to the reference state (e.g., blocked peptide), where the protein residues are fixed to some state such that the value is pH independent; the second (∆G titr (pH)) accounts for the contribution from the titratable residues in the protein as they (de)protonate with pH.