The enthalpies of proton ionization of the biochemical buffers N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES) and N-tris[hydroxymethl]methyl-2-aminoethanesulfonic acid (TES) were obtained in water-methanol mixtures in which the methanol mole fraction (X-m) varied in the range 0-0.36. For both buffers, ionization enthalpy for the first proton (DeltaH(1)) was small in all solvent media. However, upon addition of methanol, DeltaH(2) increased steadily from 22.2 to a maximum of 27.2 kJ mol(-1) for BES, whereas for TES it varied from 30.0 to 32.4, with a minimum of 28.6 kJ mol(-1) at X-m = 0.123. It is noteworthy that this solvent composition lies within the region of maximum structure enhancement of water by methanol. The results were interpreted in terms of methanol-water interactions. (C) 2003 Elsevier B.V. All rights reserved.