266 Burrellconcentrations of enzyme, RNA with secondary structures will not be
cleaved. If it is important to achieve total RNA hydrolysis either the
concentration of enzyme must be high or the ionic conditions adjusted
to ensure the RNA is single stranded.
2.4. pH and Ionic Strength
The optimum pH is in the region of 7.6 (2), although other workers
have reported different values with different sources of RNA. As indi-
cated earlier, assays are frequently done at pH 5.0 and this is usually
because higher pH values can lead to higher background and nonen-
zymic degradation. The importance of ionic strength is reviewed by
Afinsen (13) and must be carefully controlled for reproducible results.
For natural substrates an ionic strength of 0.1 gives optimal activity.
However, 0.35 is optimal for synthetic substrates (14).
At physiological pH and ionic strength, RNaseAhas very little activity
against double-stranded RNA (12) or poly A (15). However, at lower
ionic strength or as indicated in Section 2.3. at high substrate and
enzyme concentrations, these forms of RNA may be degraded (12,15).
2.5. Inhibitors
Many ions, both cations and anions, have been reported to inhibit
RNase, but the literature contains many conflicting results. It is clear
that the particular conditions of assay can alter the result. Thus Ca 2÷
ions have been reported to stimulate activity (14) and to have no effect
(16), but the conditions of assay were not the same. In general it would
appear that divalent cations can interfere with the assay and may inhibit
the reaction. Thus in critical experiments it is probably wise to avoid
high concentrations of cation, test for interference under the condi-
tions of use, and include EDTA in the reaction mixture.
The products of digestion, especially pyrimidine nucleotides, may
inhibit the reaction (14). Anionic polymers will inhibit at high concen-
trations probably by combination with the enzyme (13).
Many tissues contain proteins that inhibit RNase. Mammalian pla-
cental tissue has commonly been used as a source of this protein. It is
an acidic protein with an approximate mol mass of 50,000 Da and
forms a 1:1 complex with RNase A. The definition of a unit of inhibitor
is the amount that inhibits 5 ng of RNaseAby 50%. Reagents that react
with free thiol groups will inactivate the inhibitor.