Description
The Ribonuclease A (RNase A) is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues. It cleaves the phosphodiester bond between the 5'-ribose of a nucleotide and the phosphate group attached to the 3'-ribose of an adjacent pyrimidine nucleotide. The resulting 2', 3'-cyclic phosphate is hydrolyzed to the corresponding 3'-nucleoside phosphate (1, 2).

Source
Bovine pancreas.

Molecular Weight
13.7 kDa monomer.

Applications

• Plasmid and genomic DNA preparation (3, 4), ..
• Removal of RNA from recombinant protein preparations.
• Ribonuclease protection assays (3).
• Mapping single-base mutations in DNA or RNA (5, 6).

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and proteases confirmed by appropriate quality tests. Functionally tested for RNA digestion in a plasmid DNA purification procedure.

Concentration
10 mg/ml.
Protein concentration is determined by measuring the absorbance at 278 nm using molar absorption coefficient e=9800M^-1cm^-1 (7).

Definition of Activity Unit
One unit of the enzyme causes an increase in absorbance of 1.0 at 260 nm when yeast RNA is hydrolyzed at 37°C and pH 5.0.
Fifty units are approximately equivalent to 1 Kunitz unit (8).

Specific Activity
>5000 u/mg protein (>100 Kunitz units/mg protein).

Storage Buffer
The enzyme is supplied in: 50 mM Tris-HCl (pH 7.4) and 50% (v/v) glycerol.

Inhibition and Inactivation

• Inhibitors: the most potent inhibitor is a ~50 kDa protein from cytosol of mammalian cells, e.g., RiboLock™ Ribonuclease Inhibitor.
  Other inhibitors: uridine 2’,3’-cyclic vanadate, 5’-diphosphoadenosine 3’-phosphate and 5’-diphosphoadenosine 2’-phosphate (2), SDS, diethyl pyrocarbonate, 4 M guanidinium thiocyanate plus 0.1 M 2mercaptoethanol and heavy metal ions.
• Inactivated by phenol/chloroform extraction.

Note

• The working concentration for RNase A is 1-100 µg/ml depending on the application.
• The enzyme is active under a wide range of reaction conditions. At low salt concentrations (0 to 100 mM NaCl), RNase A cleaves single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids. However, at NaCl concentrations of 0.3 M or higher, RNase A specifically cleaves single-stranded RNA (9).

References

1. Blackburn, P., Moore S., Pancreatic ribonuclease, The Enzymes, V, (Boyer, P.D, ed.), Academic Press, New York, the third edition, vol. 15, 317-433, 1982.
2. Raines, R.T., Ribonuclease A, Chem. Rev., 98, 1045-1065, 1998.
3. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1.31-1.38, 2001.
4. Sharma, R.C., et al., A rapid procedure for isolation of RNA-free genomic DNA from mammalian cells, BioTechniques, 14, 176-178, 1993.
5. Myers R.M., et al., Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes, Science 230, 1242-1246, 1985.
6. Winter E., et al., A method to detect and characterize point mutations in transcribed genes: Amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells, Proc. Natl. Acad. Sci. USA, 82, 7575-7579, 1985.
7. Sela, M., Anfinsen, C.B., Some spectrophotometric and polarimetric experiments with ribonuclease, Biochim. Biophys. Acta, 24, 229-235, 1957.
8. Kunitz, M.A., A spectrophotometric method for the measurement of ribonuclease activity, J. Biol. Chem., 164, 563-568, 1946.
9. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.13.1, 1994-2005.