Removal of Nucleic Acids

The purification of enzymes from bacterial homogenates and to a lesser extent animal
tissue homogenates is complicated by the presence of nucleic acids. Enzymes
modified nucleic acid provide the foundation for the molecular biology technology. They
are used to degrade, join or remove portions of the nucleic acids in a controlled and
generally defined manner. These high molecular weight biopolymers increase the
viscosity of the homogenate to an unmanageable level, at least in large scale
operations where the next step in the process calls for filtration or centrifugation to
remove cell debris. Some intracellular enzymes preparation contain nucleic which can
give to increased viscosity interfering with enzymes purification procedures particularly
ultrafiltration and centrifugation to remove cell debris.

Factors such as shear, High pH, low ionic strength and the presence of endogeneous
nucleases denatures nucleic acids. The problem of high viscosity may be eliminated by
brief mechanical shearing in a Waring Blender, colloid mill or even vigorous mechanical
stirring of the cell suspension. Nucleic acids can be precipitated by high molecular
weight polyvalent cations such as protamine sulfate, cetyltrimethyl ammonium bromide
(CTAB), streptomycin sulfate and polyethyleneimine. To remove

Polyvalent cations form a complex between negatively charged Phosphate residues of

the nucleic acid molecules and positively charged groups of the precipitant. The
resulting complex is then removed by centrifugation. With extracts of E.coli it was shown
that the effectives in precipating nucleic acids decreased in the order:

POLYLYSINE> POLYETHYLENEIME> CTAB> STREPTOMYCIN SULPHATE>

PROTAMINE SULPHATE>MANGANESE CHLORIDE

In the laboratory, Protamine sulfate has been used for the precipitation of nucleic acids
from extracts of beef liver in the purification of glutamate dehydrogenase.

After measuring the protein content, Protamine sulfate solution (20 mg/mL) at pH 7.0
was added to the partially purified enzyme solution to a final concentration of 100 mg/g
protein. Approximately 60% of the nucleic acids were precipitated and removed by
filtration. Subsequent purification of the enzyme by salt precipitation also reduced the
nucleic acid content in the finished product. Welch and Scopes have purified yeast
phosphofructokinase by adsorption of the enzyme to protamine-nucleic acid
precipitates. Precipitation of proteins away from nucleic acids is possible with dextran-
Polyethylene glycol and high concentration of sodium chloride.
Other patented substances, which can be used for the precipitation of nucleic acids and
some nonactive proteins from microbial enzyme extracts are water-soluble cationic
synthetic polymers comprised of cationic monomers such as acrylamide, N-methyl-2-
aminoethylmethacrylate hydrochloride, 2-hydroxyethyl acrylate, and 2-(N,N,
trimethylammonium)-ethyl methacrylate chloride. These synthetic polymers have been
found useful in purifying enzyme extracts from a variety of microorganisms. A partial list
includes, for example, M. flavins, B. megaterium, P. testeroni, and S. faecalis.