Proteins and other large molecules are usually not available in sufficient quantities to grow crystals in the relatively large batch methods described by the other parts of this program. Additionally, these molecules are normally grown from aqueous solution using a variation of the simple "vapor diffusion" technique. Here a concentrated solution of salts, ligands and/or precipitating agent is made and used as the "outside" or "reservoir" solution that will lower the solubility of the macromolecule. A solution of the macromolecule is made at more dilute concentrations of salts, ligands and precipitating agents. This is called the "dip" solution, from which the crystals will grow. The concentration of the components that lower the solubility increases slowly by vapor diffusion. Water vapor usually travels from the more dilute macromolecular solution and enters the more concentrated reservoir solution, thus decreasing the volume of the "dip" solution and increasing the concentration of the macromolecule and precipitation agents. Once the "dip" solution reaches saturation, crystals begin to grow.
      There are four ways to reach slight supersaturation in growing protein crystals: salt, solvent, precipitant and pH. Macromolecules, such as proteins and nucleic acids, are more soluble in low concentrations of salt than they are in either pure water or high concentrations of salt. At low salt concentrations a protein-salt complex forms that is more polar than the protein alone, and thus the complex is more soluble in water, "salting in". At high salt concentrations the water molecules are more attracted to the salts than to the protein owing to the higher charge density on the small ions. As the water molecules are increasingly associated with the salt, the amount of water available to dissolve the protein is decreased and the protein is "salted out". The effect of a particular salt depends on its ionic strength, which combines both the concentration of ions and their charge. Multiply charged ions are much more effective at lowering solubility than are singly charged ions.
       The solvent method changes the dielectric constant. The electrostatic attraction is inversely proportional to the dielectric constant (Coulomb's Law). In water, with a dielectric of 80 v, electrostatic forces are weak. In organic solvents, with dielectric constants of 2-20 v, the electrostatic attraction is much greater. However, in organic solvents, one must achieve a balance between keeping the protein native (nonpolar in, polar out) and lowering the solubility by getting the proteins to attract one another.
       A precipitant such as a PEG polymer can be employed. It is most effective to select the one with a molecular weight similar to the protein of interest. The polymer competes with the protein for the water and as the water available to the protein decreases crystallization begins.
       The solubility of macromolecules is also pH dependent. The association of the molecules required for crystallization is greatest for neutral molecules. This is at the pI, where the molecule does not migrate in an electric field. Here the molecules have least repulsion for their neighbors. The pH can be slowly changed by using volatile acid (acetic acid) or base (ammonia). Unlike the methods that depend upon the diffusion of water vapor and increase the concentration of the protein, precipitation may result from a change in the pH with no change in the concentration of the protein.
      Acknowledgement: This description was provided by Professor Martha Teeter, Department of Chemistry, Boston College