Ribonuclease A (RNase A), DNase-free

Feature

The RNase A is free of DNase activity. It is not necessary to heat it before use.

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-1 cm-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).

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.