Separation of bases, ribonucleosides and deoxyribonucleosides by anion-exclusion and partition chromatography on cation-exchange resin: Application to the assay of ribonucleotide reductase, deaminase and nucleosidase
✍ Scribed by Bimal C. Pal; James D. Regan; Franklin D. Hamilton
- Publisher
- Elsevier Science
- Year
- 1975
- Tongue
- English
- Weight
- 477 KB
- Volume
- 67
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
CDP and CTP are used as substrates in the assay of ribonucleotide reductase. deaminase and nucleosidase activity in crude enzyme preparations. After incubation. the nucleotides are hydrolyzed to nucleosides by sequential treatment with potato apyrase and alkaline phosphatase. An aliquot is then chromatographed on a cation-exchange column at 50°C with 0.1 M boric acid, adjusted to pH 7.4 with ammonia, used as eluant. The pyrimidines Ura, Urd, dUrd, Cyt, Cyd and dCyd are separated and eluted in about 50 min in small volumes. Assays by this procedure of CTP reductase activity in crude fractions of ribonucleotide reductase from Euglena gracilis gave results comparable to those obtained by the standard method. The new procedure is also applicable when adenine or guanine nucleotides are used as substrates. The adenine derivatives Ade, Ado. dAdo, Hyp, Ino. dlno as well as the guanine derivatives Cua, Guo. dGuo, Xan, Xao are separated from each other in this chromatographic system in about an hour.