8 Weir
has been commercially available for at least 25 years, most of the work
on the properties of the enzyme being done in the 1960s and, to date,
no DNase has been found to replace it in the dual tasks of complete
degradation of"nuisance material" DNA and the partial hydrolysis of
DNA molecule in such techniques as nick translation.
In the following sections, the properties of DNase I are discussed in
detail with special emphasis directed toward comparing the activity of
DNase I with that of other types of DNase, most notably DNase II.
- DNase I (EC 3.1.21.1)
2.1. Reaction
Bovine pancreatic DNase, or more usually, DNase I, catalyzes the
hydrolysis of ds DNA molecules but, at high concentrations of enzyme,
ss DNA will also be digested. "Complete" hydrolysis results in the
formation of small oligonucleotide products that are resistant to fur-
ther cleavage, but are acid soluble (see section on assay of DNase
activity); cleavage results in the formation of 5' monoesterified prod-
ucts. Cleavage of DNA substrate with DNase II (spleen DNase or acid
DNase, EC 3.1.22.1), in contrast, results in the formation of 3'
monoesterified products.
2.2. pH Optimum
Possibly the major reason that DNase I is preferred to DNase II is
that DNase I has optimum activity in the region of pH 7-8, whereas
DNase II, as its alternative name describes, has a pH optimum in acidic
condition: pH 4.2-5.5.
2.3. Activators and lnhibitors
DNase I has an absolute requirement for divalent metal cations. The
most commonly used is Mg 2÷, however Mn 2÷, Ca 2+, Co 2+, and Zn 2+
will also activate DNase I. Concentrations of Mg 2÷ above approx 50
mM become inhibitory, which is not the case for Co 2÷ and Mn 2÷.
Monovalent metal ions are also inhibitors of the enzyme activity. In
the presence of Ca 2÷, Mg 2÷ has a synergistic effect, i.e., the rate of
hydrolysis of DNA in the presence of both ions is more than the sum
of the rates of hydrolysis of DNA in the presence of each ion sepa-
rately. A total of 0.1 mM Ca 2÷ is sufficient to give this enhanced reac-
tivity in the presence of 10 mM Mg 2÷, however the rate of hydrolysis
of DNA is still greatest in the presence of Mn 2+ ions (see Section 2.4.).
