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.