Proton ionization enthalpies for three structurally related biological buffers, each with two ionizable protons, were determined using solution calorimetry. These buffers are 3-[N-morpholino]propanesulfonic acid (MOPS), 4[N-morpholino]butanesulfonic acid (MOBS) and 3-[N-morpholino]-2-hydroxypropanesulfonic acid (MOPSO). Enthalpies were obtained in water-methanol mixtures with methanol mole fraction (X(m)) from 0 to 0.360. The first ionization enthalpy (Delta H(1)) of all buffers was small (+/- 1.2 kJ mol(-1)) at all solvent compositions. The second ionization enthalpy (Delta H(2)) increased steadily with X(m), rising from 20.7 to 25.6 kJ mol(-1) for MOPS, 26.3-31.0 kJ mol(-1) for MOBS and 23.5-32.2 kJ mol(-1) for MOPSO. The results were compared to those for related biological buffers. The observed variations were interpreted in terms of solvent-solvent and solvent-solute interactions. (C) 2011 Elsevier B.V. All rights reserved.