Intrinsic Hydrogen–Deuterium Exchange Rates in H 2 O/D 2 O Mixtures

: Hydrogen-deuterium exchange (HDX) measurements are widely used to probe protein structural dynamics. Quantitative interpretation of HDX data relies on the concept of an intrinsic exchange rate, which is well characterized in isotopically pure H2O or D2O but does not explicitly account for the back exchange that necessarily occurs in H2O/D2O mixtures: in this case, both the approach-to-equilibrium rate and the equilibrium deuterium enrichment of amides depend nontrivially on solvent composition and acidity. A practical method is presented to predict intrinsic forward and reverse amide exchange rates in H2O/D2O mixtures. The approach combines known second-order reference rates measured in pure solvents with established empirical descriptions of H2O/D2O mixtures. The resulting framework yields explicit expressions for forward and back exchange rates as functions of solvent composition and acidity and correctly recovers the known limits in pure H2O and pure D2O. The model predicts composition-dependent kinetic isotope effects and an equilibrium amide fractionation factor of ϕ = 1.20 for unstructured peptides under base-catalyzed conditions, in close agreement with the experimental value 1.22 reported for poly-d,l-alanine. By providing a physically motivated description of exchange in mixed solvents, this method offers a practical starting point for quantitatively correcting back exchange in HDX-MS and HDX-NMR experiments.