Methods in Molecular Biology • 16 Enzymes of Molecular Biology

(Nancy Kaufman) #1
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.


  1. 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

Free download pdf