Enzyme Technology
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, Kintercept
is the intercept constant, Ksalt is the salting out constant and T
is the ionic strength which is proportional to the concentration of a
precipitating salt. Kintercept is independent of the salt used but
depends on the pH, temperature, enzyme and the other components in the solution.
Ksalt 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.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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