High-pressure liquid chromatography-ultraviolet analysis of intracellular nucleotides

✍ Scribed by Alfonso L. Pogolotti Jr.; Daniel V. Santi

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 974 KB
• Volume: 126
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90524-3

✦ Synopsis

A high-pressure liquid chromatography-ultraviolet (HPLC-uv) system is described which resolves over 20 uv-absorbing components of cell extracts. Together with a previously described procedure for HPLC-uv analysis of deoxynucleoside triphosphates (C. Garrett and D. V. Santi, 1979, Anal. Biochem. 99,268-273), pool sizes of the major purine and pyrimidine nucleotides in cell extracts may be readily determined. The utility of this system is illustrated by comparison of HPLC-uv protiles of extracts from control and methotrexate-treated L12 10 cells; in addition to quantitation of changes in nucleotide pools, a thusfar unrecognized increase of an unidentified metabolite is reported in extracts of drug-treated cells. The chromatographic behavior of over 35 naturally occurring nucleotides in this system is described which permits tentative assignment of most of the peaks observed in HPLC-uv profiles of cell extracts. We also provide an evaluation of potential sources of error in the analysis of nucleotide pool sizes and offer suggestions as to how they may be detected and circumvented.