Concentration by precipitation

Precipitation of enzymes is a useful method of concentration and is ideal as an initial step in their purification. It can be used on a large scale and is less affected by the presence of interfering materials than any of the chromatographic methods described later.

Salting out of proteins, particularly by use of ammonium sulphate, is one of the best known and used methods of purifying and concentrating enzymes, particularly at the laboratory scale. Increases in the ionic strength of the solution cause a reduction in the repulsive effect of like charges between identical molecules of a protein. It also reduces the forces holding the solvation shell around the protein molecules. When these forces are sufficiently reduced, the protein will precipitate; hydrophobic proteins precipitating at lower salt concentrations than hydrophilic proteins. Ammonium sulphate is convenient and effective because of its high solubility, cheapness, lack of toxicity to most enzymes and its stabilizing effect on some enzymes (see Table 2.4). Its large-scale use, however, is limited as it is corrosive except with stainless steel, it forms dense solutions presenting problems to the collection of the precipitate by centrifugation, and it may release gaseous ammonia, particularly at alkaline pH. The practice of using ammonium sulphate precipitation is more straightforward than the theory. Reproducible results can only be obtained provided the protein concentration, temperature and pH are kept constant. The concentration of the salt needed to precipitate an enzyme will vary with the concentration of the enzyme. However, fractionation of protein mixtures by the stepwise increase in the ionic strength can be a very effective way of partly purifying enzymes.

The solubility of an enzyme can be described by the equation

              (2.11)

where S is the enzyme solubility, K_intercept is the intercept constant, K_salt is the salting out constant and T is the ionic strength which is proportional to the concentration of a precipitating salt. K_intercept is independent of the salt used but depends on the pH, temperature, enzyme and the other components in the solution. K_salt depends on both the enzyme required and the salt used but is largely independent of other factors. This equation (2.11) may also be used to give the minimum salt concentration necessary before enzyme will start to precipitate; the concentration change necessary to precipitate the enzyme varying according to the magnitude of the salting out constant.

Some enzymes do not survive ammonium sulphate precipitation. Other salts may be substituted but the more favoured alternative is to use organic solvents such as methanol, ethanol, propan-2-ol and acetone. These act by reducing the dielectric of the medium and consequently reducing the solubility of proteins by favouring protein-protein rather than protein-solvent interactions. Organic solvents are not widely used on a large scale because of their cost, their flammability, and the tendency of proteins to undergo rapid denaturation by these solvents if the temperature is allowed to rise much above 0°C. On safety grounds when organic solvents are used, special flameproof laboratory areas are used and temperatures maintained below their flashpoints.

Except when enzymes are presented for sale as ammonium sulphate precipitates, the precipitating salt or solvent must be removed. This may be done by dialysis, ultrafiltration or by using a desalting column of, for instance, Sephadex G-25.