The multiple dissociation constants of glutathione disulfide: interpreting experimental pH-titration curves with ab initio MD simulations

Abstract

The hexapeptide glutathione disulfide (GSSG) has six ionizable groups with six associated dissociation constants. The experimentally measured pH-titration curve, however, does not exhibit the six corresponding equivalence points and bears little resemblance to standard textbook examples of acid–base pH-titration curves. The curve highlights the difficulties in determining multiple pK_a values of polyprotic acids – typically proteins and peptides – from experiment. The six pK_a values of GSSG can, however, be estimated using Car–Parrinello molecular dynamics (CPMD) simulations in conjunction with metadynamics sampling of the underlying free energy landscape of the dissociation reactions. Ab initio MD simulations were performed on a GSSG molecule solvated by 200 water molecules. Using the estimated pK_a values the theoretical titration curve was calculated and found to be in good agreement with experiment. The results clearly highlight how dissociation constants estimated from ab initio MD simulations can facilitate the interpretation of the pH-titration curves of complex chemical and biological systems.