A new paper chromatography solvent system resolving pyrimidine-pyrimidine riboside-pyrimidine deoxyriboside mixtures
✍ Scribed by William J. Reeves Jr.; Arnold S. Seid; David M. Greenberg
- Publisher
- Elsevier Science
- Year
- 1969
- Tongue
- English
- Weight
- 186 KB
- Volume
- 30
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
During studies on deoxyribonucleoside metabolism in tissue cultures, the need arose for a paper chromatography system that could quantitatively separate pyrimidine-pyrimidine riboside-pyrimidine deoxyriboside mixtures. A search of the literature and trial experiments showed that none of the systems usually employed for the analysis of nucleic acid constituents was entirely satisfactory for this purpose. The usual solvents were incapable of resolving such mixtures, produced salt-encrusted papers, or required development at elevated temperatures, It was found that the isobutyric acid-ammonia-water solvents of Magasanik et al. (1) and of Krebs and Hems (2) could be modified to separate these substances readily by decreasing the water and ammonia content and adding toluene. The composition of the most useful modification was: isobutyric acid, 160; water, 22; 0.1 M sodium EDTA, 3; concentrated ammonia, 2; and toluene, 20 parts by volume. This new solvent was used to assay the degradation of radioisotopically labeled thymidine to thymine by Ehrlich ascites carcinoma cells in tissue culture. The experimental procedure employed and the properties of the system are described below.
Protein was precipitated from tissue culture supernates by addition of cold perchloric acid (final concentration 0.3 N) . After centrifugation, the supernates were neutralized with KOH and chilled to precipitate potassium perchlorate. Samples (volume < 0.5 ml) were applied as 1 X 3 cm bands along a horizontal line 20 cm below the upper edge of Whatman 3 MM paper (46 X 57 cm). The top of the paper was notched (Fig. 1) to reduce the wick area and thus decrease solvent flow rate. Otherwise flow rates were excessive and spots were diffuse and poorly separated.