Amide HD Exchange

The exchange rate of a peptide amide hydrogen reflects its precise and unique environment within the protein's three-dimensional structure, and there is one such hydrogen for each amino acid in the protein, except for proline. A backbone amide hydrogen can exhibit highly variable exchange rates with solvent hydrogen, with rates ranging over eight orders of magnitude in folded proteins [4]. In contrast, amide hydrogen exchange rates in peptides lacking secondary and tertiary structure vary only about 100-fold, depending primarily on neighboring amino acid side-chains [34].

The exchange kinetics of amide hydrogens can be followed by deuterium or tritium isotope labeling with exchange times ranging from seconds to days. The exchange rates of hydrogens on -OH, -SH, -NH2, -COOH, and -CONH2 groups and the amino and carboxy termini are much faster. Carbon-centered hydrogens do not exchange under normal conditions, and undergo isotope substitution only following activation by chemical treatment, such as reaction with hydroxyl radicals [35].

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