RNase A 267
Other mechanisms of inhibiting RNase activity during RNA extrac-
tion include the use of sodium dodecyl sulfate, diethyl pyrocarbonate,
and 4M guanidinium thiocyanate plus 0.1M mercaptoethanol (5).
Oxovanadium (VO 2÷) forms a stable complex with nucleotide mono-
phosphates, which competitively inhibits RNase A, but the complex
also prevents RNA translation (17). Spermine at 0.13 mM will inhibit
tRNA hydrolysis by 50% (18). It inhibits the rate of the reaction and
does not affect the K m.
RNaseAis a common contaminant ofDNase 1 preparations and can
be removed by alkylation of a histidine residue at the active site. The
procedure was developed by Zimmerman (19). Practical protocols
have been discussed by Gurney and Gurney (20).
2.6. Stability
RNase is probably one of the most stable enzymes the molecular
biologist will use. It is quite stable over a wide range of pH values below
25 °C. At 100°C the enzyme is most stable between pH 2.0 and pH 4.5. For
example heating for 30 min at 100°C and pH 3.5 will only destroy 21%
of the activity whereas 5 min at pH 9.0 will destroy more than 90% (2).
UV light (254 nm) at pH 5.0 will inactivate the enzyme probably as
a result of destruction of the disulfide bridges in the protein (21).
Chemical destruction of these disulfide bridges will also destroy activ-
ity. If the disulfide bridges are left intact by the method of denaturation
then the protein will refold quite rapidly. Enzyme activity can still be
restored if the disulfide bridges are reoxidized, but air oxidation requires
several hours (5).
- Experimental Procedures
RNase A can be used in combination with other RNases to charac-
terize RNA directly and provide sequence information. A common use
of the enzyme is to test for complimentarity between RNA:DNA hybrids
either to remove large unhybridized regions or, under appropriate reac-
tion conditions, to identify single base differences (22). In the latter
case, an RNA fragment labeled with 32p is prepared from a DNA
template of interest. This is then hybridized to test DNA in solution
and the resulting hybrid treated with RNase A. Where single base pair
mismatches occur the RNA is cleaved. Therefore, upon electrophore-
sis, more than one labeled RNA molecule is detected.