Mung-Bean Nuclease I 255
Laskowski (3) found that a temperature of 37°C improved the hydro-
lysis of native biosynthetic d(A-T)n by auto-acceleration (interpreted
as the production of a more favorable substrate, i.e., single strands,
during the course of the experiment).
2.5. Salt Optimum
Mung-bean nuclease 1 is sensitive to ionic strength; 0.025-0.050M
ammonium acetate or Tris has been found to be optimal, whereas
concentrations of 0.2-0.4M are reported to give 80-90% inhibition of
enzyme activity (1). Salt concentration can affect stability (see Sec-
tion 2.8.) and pH optimum (see Section 2.3.).
- Assay
The most common assay for mung-bean nuclease 1 (and that which
is used by those commercial companies who supply the enzyme) is
based on the ability of the enzyme to form acid-soluble products, i.e.,
the ability to convert denatured DNA to mono- and oligonucleotides
that are not precipitated on addition of TCA, but that do contribute to
the hyperchromicity of the supernatant. Another assay, which is based
on the absolute requirement of the enzyme for Zn 2÷, involves the use
of electrophoresis. Although it is only qualitative, it does have the
advantage of being very sensitive, thus only requiring minimal amounts.
Heat-denatured DNA is incubated with the enzyme in the presence of
Zn 2÷, in the absence of Zn 2÷, and in the absence of Zn 2÷, but in the
presence of 0.001M EDTA. These DNA samples are then run on a
0.8% agarose gel (100 ng/well is sufficient) and stained with ethidium
bromide. The three lanes on the gel should show: complete breakdown
of DNA in lane 1 (optimum conditions for the enzyme); no breakdown
in lane two (although some breakdown may occur, since the enzyme
may lose only 70-80% of its original activity on removal of exogenous
Zn 2÷ [9]); and no breakdown in lane three (since 0.001M EDTA will
remove the zinc metalloportion of the enzyme, thereby inhibiting it
irreversibly). This last gel lane is important to determine the complete
absence of other DNases, since it is known that simple addition of
EDTA to an assay mixture will not necessarily protect it from all
DNase attack (11). This assay may be used with serial dilutions of the
enzyme to give an indication of specific activity.
