Method to estimate amide base-catalyzed back exchange rates in H₂O/D₂O mixtures

Measurements of hydrogen deuterium exchange (HDX) in proteins provide means to characterize protein structural dynamics. The LinderstrØm-Lang describes exchange in isotopically pure solvents, but does not account for the back exchange occurring in H 2 O/D 2 O mixtures used in experiments. "Intrinsic" exchange rates of amides maximally exposed to solvent have been measured in pure solvents, but systematic investigations in mixtures are lacking. Here, a method to predict intrinsic forward and back HDX rates in mixtures is described for the base-catalyzed pathway, which dominates near neutral conditions. The model combines literature second-order reference rates in pure solvents with the empirical dependence of the mixture ionic product of and with the probability of reprotonation by H versus D solvent species. It recovers the known rates for pure solvents, predicts composition-dependent kinetic isotope effects, and yields an equilibrium fractionation factor φ=1.20 for unstructured peptides (in close agreement with the reported PDLA value 1.22). Because it provides explicit forward and back rates as functions of solvent composition and acidity, the method offers a practical starting point for quantitatively correcting back exchange in HDX-MS and HDX-NMR workflows.