Section 12.1.3.7. Effect of concentration, time of soak and temperature on heavy-atom binding

Most heavy-atom derivatives are prepared by diffusing or dialysing the compound into the crystals. Concentrations have typically ranged from 0.1–100.0 mM. Occasionally, concentrations as low as 0.001 mM have been employed to maintain crystal integrity. Low concentrations favour sites where the interactions between the heavy atom and the protein ligands are strongest. Decreasing the number of non-specific interactions minimizes the amount of heavy-atom reagent in the lattice. The latter absorbs X-rays without contributing to the diffraction pattern except at low angles. Increasing the concentration may give rise to other binding site(s). Usually, the higher the concentration employed, the shorter the soak time required for equivalent substitution. Short soak times at high concentrations tend to denature the crystals more often than long soaks at low concentrations. At very high concentrations (i.e. > 100 mM), the heavy-atom compound perturbs the protein crystal–mother liquor equilibrium by withdrawing water molecules from the hydration shell around the periphery of the crystal. Disorder of the crystals can sometimes be avoided by the application of a cross-linking reagent (e.g. glutaraldehyde). The optimal concentration is the lowest concentration that consistently reproduces intensity differences in the diffraction pattern of 15–25% without cracking and disordering the crystals.

Length of soak may be important. The heavy-atom data bank shows that, typically, soak times range from one day to one week. Useful derivatives have been prepared with a soak time of an hour to over a year. If no binding is apparent after several days, extending the soak time to over a week may produce some binding, but this is rare. Soaks of 24 hours for simple inorganic salts and up to one week for other types of heavy-atom compounds will normally suffice when screening for binding. The concentration of the heavy-metal compound that can be achieved will depend on its solubility in the crystal stabilization solution. Normally, the longer the soak, the greater the occupancy. Exceptions can arise due to undesirable chemical reactions between components present in the derivatization solution.

For covalent-bond formation, the length of soak and the concentration can be short (e.g. 1 h, 0.01 mM). This is especially true for mercury derivatives of proteins that have reactive sulfhydryls (Ringe et al., 1983).

Variations in the temperature can also alter the rate of reaction. The UO₂ acetate derivative of rhombohedral insulin binds twenty times more slowly at 4 °C than at ambient temperature (Blundell, 1968). A lower temperature allows greater control over the rate of substitution. Conversely, heavy-atom derivatives that do not appear to bind may do so upon elevation of the temperature.