Protein hydrogen exchange studied by the fragment separation method

The potential of hydrogen-exchange studies for providing detailed information on protein structure and structural dynamics has not yet been realized, largely because of the continuing inability to correlate measured exchange behavior with the parts of a protein that generate that behavior. A promising approach to this problem was developed in which tritium label at exchangeable proton sites can be located by fragmenting the protein, separating the fragments, and measuring the label carried by each fragment. However, severe losses of tritium label during the fragment separation steps have so far rendered the results ambiguous. This paper describes methods that minimize losses of tritium label during the fragment separation steps and correct for losses that do occur so that the label can be unambiguously located and even quantified. Steps that promote adequate fragment isolation are also described