264 Burrell
nus destroys enzyme activity. There is a considerable literature on the
folding of the RNase A polypeptide and the importance of different resi-
dues in this folding (5).
Pancreatic RNases from many species are glycosylated (6). There
are four attachment sites for the glycosidic side chains and all involve
asparagine residues. It has been suggested that the role of these side
chains is to protect the RNase molecule from being absorbed in the gut
so that it is transported to the large intestine where it would then be
available to hydrolyze the RNA from the cecal microflora. In vivo RNase
A protein is first synthesized as a 16,100 Da protein that contains a 25
amino acid extension at the N-terminus, which is removed on secretion.
It appears that the presecretory form is also enzymatically active (7).
2.1. The Reaction
RNase A is an endoribonuclease and therefore will cleave RNA to
both mononucleotides and oligonucleotides. The enzyme attacks at
the 3' phosphate of a pyrimidine nucleotide. Therefore under the ap-
propriate reaction conditions the sequence pG-pG-pC-pA-pG will be
cleaved to give pG-pG-pCp and A-pG. A cyclic intermediate is formed
(see Fig. 1). This specificity for pyrimidine residues should be consid-
ered when using RNase A in protocols, such as the one in this chapter,
for detecting mismatches. Thus, mismatches involving pyrimidines
are likely to be cleaved preferentially to those involving purines unless
conditions are adjusted appropriately (see Sections 2.3. and 2.4.).
2.2. Assay and Unit Definition
There are several different assays and units of activity used for
RNase A. As with all enzyme assays, the purity of the substrate will
affect the result. Kunitz (2) was the first to crystallize the protein and
used an assay that depended on a fixed starting amount of yeast nucleic
acid (0.5 mg total phosphate in 1 mL of 0.1M acetate buffer at pH 5.0).
After 10 min incubation at 25°C the RNA is precipitated with 2 mL of
0.25% uranium acetate and the solution filtered. The filtrate is then
assayed for total phosphate. A unit of activity for this assay is defined
as the activity that produces 1 × 10 -3 mg of soluble phosphorus per mL
of digestion mixture.
An alternative assay, which depends on the shift in the absorption spec-
trum of RNA on digestion with RNase, was introduced by Kunitz in 1946
(8). RNA digestion causes a decrease in the absorption at 300 nm. Kunitz