254 Sharp and Slater
- Enzyme Data
2.1. Purification
Purification of the nuclease activity from mung-bean sprouts has
been reported by several authors. Sung and Laskowski (1) published
their original paper in 1962, where they described the enzyme as the
first peak of a series of five peaks eluting from a DEAE-Sephadex
column. The column itself was the final step in the purification pro-
cess, which gave a 2000-fold purification with a 0.5% yield. This was
followed by an improved technique in 1968 by Johnson and Laskowski
(7). Ardelt and Laskowski (2) used an eight-step process that gave a
25,000-fold purification with a yield of 20% and claimed to have
purified the enzyme to homogeneity.
2.2. Molecular Mass
The mol mass of the nuclease is between 35,000 (8) and 39,000 Da
(9) as determined by SDS gel electrophoresis. The enzyme is known
to be a glycoprotein that is 29% carbohydrate by weight and is also
reported to be composed of two subunits (25,000 and 15,000 Da)
linked by a disulfide bond(s) (4).
2.3. pH Optimum
Mung-bean nuclease has a pH optimum of 5.0, although this can be
varied (4.8-5.5) depending on reaction conditions or in order to enhance
certain characteristics of the enzyme over others. Although predomi-
nantly single-strand specific, mung-bean nuclease 1 will degrade duplex
DNA to mononucleotides at high concentrations (4). Both the hydro-
lysis of single-stranded and the hydrolysis of double-stranded DNA
have the same pH optima. The preference for single-stranded struc-
tures over double-stranded increases as the pH increases. At pH values
close to 7.0, the preference for super-coiled DNA over the relaxed
form is 30,000-fold. Increasing salt concentrations cause the pH opti-
mum to become more acidic.
2.4. Temperature
Mung-bean nuclease 1 is usually assayed and used at 37°C. Ghangas
and Wu (10) found that "terminally directed" nuclease activity decreased
at 5°C, whereas endonucleolytic "nicking" still occurred. Johnson and