242 Gray and Luwhere Vto t is the total volume of the acidified reaction mixturealiquot
in mL (Vto t = 1.24 mL if the above volumes are used). Division by the
volume of enzyme solution per aliquot (0.04 mL in the above) yields the
U/mL of nuclease in the solution before dilution into the assay mixture.
For the removal of aliquots at 5-min intervals, AA260/t should be at
least 0.02 min -l, which implies that the enzyme solution being assayed
(before dilution into the aforementioned reaction mixture) should be
in the range of 20 U/mL. Correspondingly, longer assays are needed
for accurate results with more dilute nuclease solutions. Only when
the absorbance of the acid-soluble released DNA nucleotides signifi-
cantly exceeds 1 has nonlinearity in the plots of A260 vs t been observed.
3.3. Characterization of the Duplex
Exonuclease Activity
3.3.1. Kinetics of Molecular Weight Reduction
Testing of each batch of commercial BAL 31 nuclease for its ability
to catalyze the duplex exonuclease reaction, where this use is to be
made of the enzyme, is necessary as discussed in Section 3.1. This is
conveniently done by subjecting restriction enzyme-generated frag-
ments of a relevant unique sequence (e.g., a plasmid DNA or the
cloned, isolated DNA fragment that is itself to be treated) to progres-
sive digestion and analyzing the partially degraded fragments by aga-
rose gel electrophoresis using (usually) the nondegraded restriction
fragments as molecular size markers. It is advisable to assume that the
kinetics will be those of the S nuclease, since this species usually
predominates in culture supernatants, and to adjust the amount of
enzyme accordingly if significantly faster degradation is observed.
For estimation of the rate of degradation, the equation:
v0][S ] -- Vmar app ] [S] -t- gm app (2)
has been used (6), where v 0 is the initial reaction velocity, IS] is the
substrate concentration (mol/L of duplex termini), Vmax app is the
apparant maximum velocity corresponding to the concentration of
nuclease used, and gm app is the apparent Michaelis constant. It is
important to note that the kinetic parameters are apparent values and
do not necessarily have the interpretations associated with Michaelis-
Menten kinetic analysis of reactions in which only a single enzyme-
substrate need be considered. At least two such intermediates, one