文摘
We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine C蔚1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second-order with respect to the concentrations of the imidazolium cation and OD鈥?/sup> in D2O. The second-order rate constant (k2) of this reaction was calculated from the pH dependency of the pseudo-first-order rate constant (k蠁) obtained from the change in average mass [螖Mr (0 鈮?螖Mr < 1)] of a peptide fragment containing a defined histidine residue at incubation time (t) such that k蠁 = 鈭抂ln(1 鈥?螖Mr)]/t. We preferred using k2 rather than k蠁 because k2max (maximal value of k2) was empirically related to pKa as illustrated with a Br酶nsted plot [log k2max = 鈭?.7pKa + 伪 (伪 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(k2max/k2) much larger than that of His119 upon binding with cytidine 3鈥?monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, we need to consider the hydrogen bonds of the imidazole group with nondissociable groups to interpret an extremely slow H/D exchange rate of His48 in a partially solvent-exposed situation.