Ribonuclease composition

Fig. 2.—A Portion of the Proton-decoupled, Natural-abundance, 13C-N.m.r. Spectra of Model Compound 6 and Bovine Ribonuclease B at 67.9 MHz. [(A) Compound 8 in HzO (25 mM, pH 6.5) after 8192 scans (2-s recycle-time) (B) spectrum of ribonuclease B after digital subtraction of the spectrum of ribonuclease A. (This enzyme has the same amino acid composition as ribonuclease B, but contains no carbohydrate.) Spectra were taken from Ref. 27.1...

Ribonuclease T2 (5) was found in 1957 by Sato and Egami in Taka-diastase (12). Since the partially purified RNase T2 was found to preferentially attack phosphodiester bonds of adenosine-3 -phosphate in RNA (80), this enzyme had long been expected to be specific for adenylic acid phosphodiester bond if it could be completely purified. However, sufficiently purified RNase T2 showed no absolute base specificity (81, 82) and was found rather to split all phosphodiester bonds in RNA with a preference for adenylic acid bonds. Thus, RNase T2 is effective in the analysis of the base composition of RNA. 

CHW Hirs, WH Stein, S Moore. The amino acid composition of ribonuclease. J Biol Chem 211 941-949, 1954. 

As an example of the use of paper electrophoresis for the fractionation of oligonucleotides consider the fractionation of some decanucleotides obtained from MS2 viral RNA by T1 ribonuclease digestion. The specificity of the enzyme ensured that each oligonucleotide contained only one Gp residue and the decanucleotides were isolated by column chromatography ( 5.2.1.2). The oligonucleotide mixture was electrophoresed for 17 hr at 6 volt/cm in 0.02 M ammonia formate pH 2.7. At this pH the nucleotides, Ap, Cp, Gp, Up have net charges, q, of —0.08, —0.02, —0.66, 0.98 respectively. Thus the mobility of these decanucleotides should depend mostly on the ratio of Ap+Cp to Up in their composition. Good separation in the predicted order has been obtained (Rushizky et al. 1965). 

These two columns, DEAE-cellulose at neutral pH followed by DEAE-Sephadex at pH 2.7, suffice to separate all the components with different nucleotide compositions present in complete pancreatic ribonuclease digests of RNA molecules with at least about 10 nucleotides before digestion. Sometimes, these fractions contain only... 

The DEAE-Sephadex pH 2.7 fractionation gives reproducible elution positions for oligonucleotides, and the nucleotide compositions of an oligonucleotide from a pancreatic ribonuclease digest can sometimes be deduced from its relative position on the chromatogram. 

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