Ribonuclease snake venom

The question of enzyme specificity for irradiated polynucleotides is taken up in more detail in the recent review of Johns.11 The specificities of four enzymes, spleen phosphodiesterase, snake venom phosphodiesterase, pancreatic ribonuclease, and pancreatic deoxyribonuclease are discussed. 

The first RNA molecule to be sequenced was not a virus, but a tRNA. Two enzymes were required in these analyses (i) bovine pancreatic ribonuclease, which cleaved after pyrimidines (C or U), became a classic system for scientific studies after Armour Co., the hotdog Company, purified 1 kg of the enzyme and distributed it to scientists (source Wikipedia) and (ii) takadiastase ribonuclease Tl, which cleaved 3 to a guanosine (G). Each of these small fragments was further analyzed by partial digestion with snake venom diesterase from the 3 -ends. Once again, biochemistry and enzymology led to breakthroughs in chemical analysis. 

After three years of work, Holley and his associates managed to collect 1 g of alanine transfer RNA, which they used to study the sequence of the polynucleotide. To perform this task, investigators used three different enzymes pancreatic ribonuclease, taka-diastase (Tl), and snake venom phosphodiesterase. 

Fig. 26. Cleavage of ribonucleic acid by ribonuclease (vertical lines labeled R) and snake venom 5 -phosphodiesterase (broken lines labeled V). P represents phosphate linking 3 and 5 positions of adjacent nucleotides.

Compounds are isolated by preparative paper chromatography on Whatman No. 3 MM paper. Chromatograms are developed in freshly made n-butanol/water/glacial acetic acid (5 3 2) as solvent system for 2-3 days. Although this system is very slow, a more convenient system was not found because of the alkaline lability of the compounds. Purity of isolated products can be checked by paper chromatography in 1 M ammonium acetate (pH 7.4)/ethanol (1 1) and by paper electrophoresis in 0.05 M potassium phosphate at pH 7.0. Analysis of oligonucleotides is best carried out by enzymic degradation with snake venom phosphodiesterase or ribonuclease Tg. ... 

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