Section 12.1.5.5. Hydrophobic heavy-atom reagents

Since many heavy-atom reagents are hydrophilic, most interactions occur at the protein surface. However, substitution, addition or removal of the non-heavy-atom component(s) of the reagent can alter the hydrophilic–hydrophobic balance and lead to penetration of the core. For example, anionic complexes such as and are hydrophilic and would not normally enter the protein core, although organometallics, such as RHgCl and R₃PbCl (R = aliphatic or aromatic), are much more hydrophobic and can do so.

Hydrophobic organomercury compounds of the general formula RHgX, where R is an aliphatic or aromatic organic group, react with sulfhydryls through displacement of X. When X is , or , the bond is ionic, making the formation of the cation RHg⁺ easier. R is often chosen to be a small aliphatic group (e.g. CH₃, C₂H₅). However, the presence of a benzene ring enhances the stability of the heavy-atom reagent. Careful selection of the X group can assist penetration into the hydrophobic core. The hydrophobicity of X follows the order RHgR (R = aliphatic or aromatic) compounds also bind sulfhydryl residues in hydrophobic regions. The mechanism of reaction of methylphenylmercury with buried sulfhydryl groups may involve fast dissolution in the hydrophobic interior of the protein followed by a slow reaction with neighbouring sulfhydryl residues (Abraham et al., 1983). They are difficult to prepare in aqueous solutions; an aprotic solvent, such as acetonitrile, can improve solubility, but this is not normally a problem in high concentrations of organic components, such as PEG, MPD or ethanol.

Inert gases were first used in the analysis of myoglobin. Schoenborn et al. (1965) discovered that the hydrophobic site that bound also bound a xenon atom at 2.5 atmospheres. They proposed that this may be a general way of producing heavy-atom derivatives of proteins. Recently, there has been increasing interest in this idea, which has now been developed to produce well defined derivatives of a wide range of different proteins. Crystals are subjected to high gas pressures. Xenon requires about 10 atmospheres in order to get saturated binding sites. Krypton binds much less strongly and requires around 60 atmospheres. Since the binding of both inert gases is reversible, it is necessary to keep the protein crystals in a gaseous environment in a specialized pressure cell. Such pressure cells have been developed by Schiltz (1997) at LURE. Xenon binds to hydrophobic cavities, with little conformational change and a retention of isomorphism in crystals. Krypton binds at the same sites as xenon, but since it is lighter and needs higher pressure it has been exploited less by protein crystallographers. However, it has a well defined K edge at around 1 Å and so has attractions for multiple-wavelength anomalous dispersion.