Imidazole C-2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Abstract

We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine Cε1 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 of 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 Brønsted plot: logk2max=-0.7normalpKa+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 much larger change in log(k2max/k2) 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.

Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies

“…We also compared the susceptibility of each His residue (Dr) with the second-order rate constant k 2 of the H/D exchange reaction as another index of photo-oxidation, expecting that both of these parameters can have a positive correlation with the accessibility of each His residue to solvent 21 . Fig.…”

“…The pK a value and the second-order rate constant k 2 of the hydrogen/deuterium (H/D) exchange reaction at the imidazole C2 (histidine C ε1 ) position of each His residue were measured by the mass spectrometric titration of pseudo-first-order rate constant ϕ k of the H/D-change reaction against pH as reported in our previous publications 6,17,21 . The D 2 O solutions of buffers used were 50 mM sodium acetate (pH 3.5-4.5), 50 mM MES (pH 5.0-7.0), and 50 mM HEPES (pH 7.5-9.5).…”

Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen–Deuterium Exchange Mass Spectrometry

“…In this case, when the p K a values are equal, the higher k max values for His 52 and His 80 compared to His 66 , His 69 , and His 84 must be due to differences in the local protein environment, for example, in solvent accessibility or hydrogen bonding patterns. 5,14 …”

“…Previously, the uptake has been derived either from the ratio of the monoisotopic (I) and I + 1 peak 4 or by calculating the average mass from the isotopic distribution, where the difference from the unexchanged average mass is used to quantify the uptake of deuterium. 14 The first method has the disadvantage that it cannot be used in the cases of peptides containing two or more histidines. We also found that using the average mass was susceptible to artifacts due to the lowest-intensity peaks’ being unreliably detected.…”

Single-crystal time-of-flight neutron Laue methods: application to manganese catalase from Thermus thermophilus HB27

“…H/D exchanges at CE1 of the imidazole ring have been observed in histidine itself, in peptides and in proteins, and were utilized to estimate the pK a of the histidine residues. It has been suggested that the rate of exchange depends on the pH of the solution, solvent accessibility and the pK a of the histidine side chains (Bradbury et al, 1973;Hayashi et al, 2014). The exchange of the HE1 atom with DE1 has been reported for the neutron crystallographic analysis of myoglobin by Ostermann et al (2002).…”