Imidazole C-2 hydrogen/deuterium exchange reaction at histidine for probing protein structure and function with matrix-assisted laser desorption ionization mass spectrometry.

We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine C hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second order with respect to the concentrations of the imidazolium cation and OD in DO. The second-order rate constant () of this reaction was calculated from the pH-dependency of the pseudo-first-order rate constant () obtained from the change of average mass Δ (0 ≤ Δ < 1) of a peptide fragment containing a defined histidine residue at incubation time () such that = − [ln(1−Δ)]/. We preferred using rather than because (maximal value of ) was empirically related to p as illustrated with a Brønsted plot: ( is an arbitrary constant), so that we could analyze the effect of structure on the H/D-exchange rate in terms of representing the deviation of from . In the catalytic site of bovine ribonuclease A, His12 showed much larger change in compared with His119 upon binding with cytidine 3′-monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, there is a need of considering the hydrogen bonds of the imidazole group with non-dissociable groups to interpret an extremely slow H/D exchange rate of His48 in partially solvent-exposed situation.