Estimation of [ 32P]deoxyribonucleoside triphosphates in cell extracts using periodate treatment

• Published in: Analytical biochemistry Vol. 82; no. 2; pp. 591 - 595
• Main Authors: Reynolds, Eric C., Finch, Lloyd R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.1977
• Subjects: Cell Line; Chromatography, Thin Layer - methods; Deoxyribonucleotides - analysis; Oxidation-Reduction; Periodic Acid
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(77)90200-7

Radioisotopic labeling of nucleotides in cells before extraction, coupled with two-dimensional chromatography on PEI-cellulose 1 1 Abbreviations: dNTP, deoxyribonucleoside triphosphate(s); rNTP, ribonucleoside triphosphate(s); PEI, polyethyleneimine. thin layers (1,2), has been successfully used in the estimation of rNTP, ADP, and GDP (3,4). This procedure has however been less successful for the dNTP, because the large amounts of rNTP tend to overlap the dNTP so that no system for the two-dimensional development of the chromatograms has been found to give consistently satisfactory separation of all four dNTP from contaminating rNTP. Yegian (5) overcame this difficulty by an initial oxidation of the ribonucleoside compounds in the extract with periodate followed by a fairly complex one-dimensional chromatographic procedure to isolate the dNTP. We have used the initial oxidation with periodate followed by the two-dimensional procedure referred to above (1) and applied the method successfully to extracts of bacterial and mammalian cells in liquid culture.