240 Gray and Luexonucleolytic attack from the 5' ends, indicate that 90% of Vmax app for
the S and F nucleases (10) can be obtained with approx 34 and 9 lag/
mL, respectively, of a DNAthe size of ~X 174. Since the denatured calf
thymus DNA normally used in this assay is not expected to be larger
in molecular weight that ~X174 DNA and is routinely used at
concentrations of at least several hundred lag/mL, the concentration of
ends should be much more than sufficient to provide maximum velocity
conditions. Data with calf thymus DNA as substrate in the velocity vs
substrate concentration curve mentioned above (6) showed that VmJ pp
was reached at concentrations near 30 ~tg/mL, which supports the
notion that the standard assay provides maximum velocity conditions.
However, this assay cannot be used to predict the kinetics of the duplex
exonuclease reaction for mixtures of the two forms.
The use of duplex DNA to assay the nuclease through the release of
nucleotides owing to the duplex exonuclease activity, as recommended
by some suppliers, will not take place under close to Vma x conditions
unless short duplexes are used at high concentrations. For a DNA of
1000 bp, its concentration would have to be over 150 ~tg/mL for the F
nuclease to achieve 90% of maximum velocity according to the most
recent estimates for the apparent K,n (10); for the S nuclease, the cor-
responding concentration would be in the 2 mg/mL range. DNA of
defined molecular size is required so that the substrate concentration
(molar concentration of termini) is known. If enzyme is assayed using
duplex DNA for the purpose of determining the activity of a particular
batch on linear duplexes, care should be taken to keep the substrate
concentration in subsequent digests close to those in the pilot experi-
ments. Also, the G + C content of the DNA in the pilot experiments
should not differ significantly from that of DNA in experiments based
on the pilot studies, unless the F nuclease is used (see Section 3.3.1 .).
Finally, there is a DNA length dependence of the kinetic parameters.
(see Section 3.3.3.)
3.2. Assay Using Single-Stranded DNA
This assay has been described in detail (4), but is reproduced here
for the convenience of the reader and because of recent modifications
(7) in the procedure. Calf thymus DNA (Type I, Sigma Chemical Co.,
St. Louis, MO) is dissolved at a nominal concentration of 1-2 mg/mL
in BE buffer (100 mM NaC1, 20 mM Tris-HC1, 1 mM EDTA [pH 8])