The solubility of a macromolecule is a complex function of a number of factors, such as pH, temperature, the chemical and physical nature of the macromolecular surface, and the nature of the precipitant (or solubilizer) used. The purposes of precipitating proteins out of solution may be varied.
1. During the purification, preparation of a particular protein out of a tissue extract or cell homogenate, the approach can be that of partial precipitation, or fractionation by precipitation using mild nondenaturing precipitants. This uses the solubility properties of the various components of the mixture to remove those that are less soluble than the desired product, or to separate the product in precipitated form from more soluble impurities. Once the more soluble and less soluble components of the original mixture have been removed, the desired protein can be purified further by cutting sharper fractions, based on solubility in a given agent, say, Na 2SO4, until only a single component is left that precipitates very sharply at a constant very narrow concentration range of the precipitant. This represents pure protein by the criterion on solubility (1).
2. Proteins may be precipitated out of solution in the form of crystals for the purpose of X-ray crystallography structural studies. This involves very special techniques (see Crystallization).
3. At times, when it is necessary to obtain a protein-free solution for the purpose of analysis for other components, in which proteins may interfere, proteins are precipitated in a denatured, generally coagulated state. This is done by the addition of general strong precipitants, with trichloroacetic acid being an additive of choice.
Precipitation of proteins during purification or crystallization is usually accomplished by the gradual addition at high concentration (0.3 to 10M) of small molecules that interact weakly, and gently, with the proteins. All these are known to be preferentially excluded from the protein accessible surface at the environmental conditions (such as temperature) used (2) (see Preferential Hydration). Many of them are structure stabilizers (see Stabilization And Destabilization By Co-Solvents). The most common precipitants are sulfate salts (ammonium sulfate, sodium sulfate, magnesium sulfate); ammonium sulfate is the most commonly used precipitant because of its high solubility in water and strong salting out properties (see Salting In, Salting Out).
Some organic solvents can also be used as precipitants (3). These include ethanol, acetone, and butanol. They must be used, however, at low concentration (about 5 to 10%) since they tend to induce protein denaturation. Two organic compounds, 2-methyl-2,4-pentanediol (MPD) and polyethylene glycol (PEG), have been found to be excellent protein precipitants and good crystallizing agents. They may be used at a high concentration (eg, 50% MPD), but their use is limited to room temperature at which they are preferentially excluded from proteins. At higher temperatures, however, both become good protein denaturing agents.
