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 Ce1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second-order with respect to the concentrations of the imidazolium cation and OD– in D2O. The second-order rate constant (k2) of this reaction was calculated from the pH dependency of the pseudo-first-order rate constant (kφ) obtained from the change in average mass [ΔMr (0 ≤ ΔMr < 1)] of a peptide fragment containing a defined histidine residue at incubation time (t) such that kφ = −[ln(1 – ΔMr)]/t. We preferred using k2 rather than kφ because k2max (maximal value of k2) was empirically related to pKa as illustrated with a Bronsted plot [log k2max = −0.7pKa + α (α is an arbitrary constant)], so that we could analyze the effect of structure on the H/D exchange rate in terms of log(k2max/k2) representing the deviation of k2 from k2max. In the catalytic site of bovine ribonuclease A, His12 showed a change in log(k2...