BAL 31 Nucleases 239
the huclease with increasing extent of exposure to protease (7). This indi-
cates a resistance of the breakup of the secondary and tertiary structure of
the S nuclease to extreme denaturing conditions that is apparently without
parallel. It is interesting that the catalytic activity is maintained near
room temperature in the presence of strong denaturants, but is rather
thermolabile. A relatively thermally sensitive binding/catalytic site(s)
in an otherwise extremely stable protein structure is suggested.
- Experimental Procedures for Unit Assay
and Characterization
of the Duplex Exonuclease Activity
3.1. General Considerations
Most commercial preparations of BAL 31 nuclease are mixtures of
the F and S forms. A problem with their use for the most common
application, the controlled length reduction of duplex DNA, is the
possible variance of the relative amounts of F and S nucleases from
batch to batch. This is because the F enzyme is derived from a larger
precursor by proteolysis, mostly in the culture supernatant, and itself
is the source of the smaller S enzyme (7). Hence, the relative amounts
of the two species can vary depending on the extent of exposure to the
supernatant protease(s) before the nucleases are separated from these.
Only one supplier (International Biotechnologies, Inc., New Haven, CT)
offers the purified S and F nucleases, which could be expected to be
reproducible in duplex exonuclease activity from batch to batch. Since
the nuclease is very stable on extended storage in the cold (4), it seems
advisable to obtain as large a sample as possible and to characterize it
for duplex exonuclease activity unless the pure S or F species is obtained.
The recommended unit assay for general characterization of a sample
(e.g., in order to check its activity after long periods of storage) is using
single-stranded DNA (4) because maximum velocity conditions, desir-
able in any enzyme assay, are readily achieved. Examination of the
velocity vs substrate concentration profile with viral ~X 174 DNA as
substrate shows that 90% of the apparent maximum velocity Vmax app
is achieved at a concentration of this single-stranded DNA near 10 ~tg/
mL for the S nuclease and at an even lower concentration for the F
nuclease (6). Calculations using recent carefully determined values
for the apparent kinetic parameters, which take into account the fact
that the major mode of degradation of single-stranded DNA is
