Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry

Abstract

An exceedingly simple and convenient method is described for measuring the hydrogen-deuterium exchange behavior of peptide bond-containing molecules by ultraviolet spectrophotometry. The exchange reaction is initiated by diluting a sample from H 2O into D 2O, or the reverse, and can be followed by an easily observable optical density change in the region of peptide absorbance. The method, unlike infrared and magnetic resonance approaches, requires only small amounts of material and, unlike the tritium-Sephadex method, is not restricted to the study of large molecules. Calibrations are provided for exchange rate as a function of pD and temperature and for the change in absorbance per mole peptide group. With this information, the exchange curve to be expected for any peptide group exposed to solvent can be predicted. Comparison with the measured data can then identify peptide-group hydrogen bonding and can also give a measure of the stability of the hydrogen-bonded structure.