Restriction Enzymes 125cysteine residues), 0.01-0.1% (w/v) Triton X-100, Tween, Lubrol, or
other detergents, as well as 0.1 mg/mL nuclease-free bovine serum
albumin (to prevent aggregation and precipitation).
Restriction enzymes should be stored in unfrozen solution at tem-
peratures below 0°C, preferably at-20°C in a 60% glycerol solution.
If stored frozen at -70°C, repeated thawing and freezing should be
avoided (the heat generated during the defrost cycles of frost-free
freezers may adversely affect the stability of a restriction enzyme
preparation!)(/75,176).
3.2. Definition of Specific Activity
The standard activity measurement for restriction enzyme prepara-
tions is based on the determination of the minimum amount of enzyme
required for the complete digestion of a standard DNA substrate, such
as bacteriophage ~, DNA. Some restriction enzymes require ~ DNA
isolated from dam or dcm E. coli strains, ~ DNA fragments, or other
DNA substrates. It must be emphasized that the activity of a given
restriction enzyme is dependent on the substrate, mainly because of
the influence of sequences flanking the recognition site. This is par-
ticularly evident with many restriction enzymes that recognize sites
composed of GC base pairs, such as NaeI, NarI, SaclI, and XmalI.
NaeI, for example, is known to cleave pBR322 DNA at positions 401,
769, and 946 more than 10 times faster than at position 1283, which
is cleaved as slowly as the single site in ~, DNA. With a given DNA
substrate, the rate of cleavage may depend on the topological state:
EcoRI, for example, attacks supercoiled pBR322 DNA more slowly
than linear or open circular BR322 DNA (99).
One unit of a given restriction enzyme is defined as the activity that
cleaves 1 ~tg of ~, DNA in 1 h under optimum buffer conditions at
(normally) 37°C. A considerable number of enzymes have abnormal
temperature optima, e.g., 25°C: SmaI, BclI (at pH 8.5); 30°C: ApaI;
55°C: BspMII, BclI (at pH 7.5); 65°C: BspMI, TaqI. Volume activity
refers to the concentration of the enzyme, i.e., U/~L.
Many restriction enzymes show a concentration-dependent change
in specificity: Loss of activity at low enzyme concentrations may be
owing to adsorption of protein at the wall of the test tube. At high
concentration, aggregation may be responsible for an apparent lower
activity. For one restriction enzyme, EcoRII, a dependence of the