Hydrogen exchange protein preparation

In an HX experiment, the sample is the protein of interest in solution. Hydrogen exchange is carried out by dilution in deuterated buffers. Proteins that are amenable to lyophilization (do not lose their sffuctural and functional properties upon lyophilization) can alternatively be lyophilized and reconstituted in deuterated buffers at the pH of interest. Sample preparation refers to the steps... 

If the carbonyl group of the molecule is considered, one can assume that, either it interacts by exchanging hydrogen bonds with its target protein or it plays an architectural role. This latter is due to the planar structure imposed by the sp character of its carbon atom. In order to decide between the two alternatives, some key substances have to be prepared and tested (Figure 19.12). 

Slow hydrogen-deuterium exchange (see Chapter 11) of native -lactoglobulin in DjO, pD 1.0 (corrected to 1.4) allowed the amide II band ( 1550 cm ) to remain and be seen in one sample of the lactoglobulin (Fig. 10.18, top curve) which was run immediately after preparation. The presence of this band shows that the native protein must be quite compact. No such bands are observed in denatured lactoglobulin or in a -casein, which have loose randomly folded structures. 

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