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The rapid separation of nucleotides in cell extracts using high-pressure liquid chromatography

Authors
Journal
Journal of Chromatography A
0021-9673
Publisher
Elsevier
Publication Date
Volume
52
Identifiers
DOI: 10.1016/s0021-9673(01)96573-2
Disciplines
  • Biology
  • Chemistry

Abstract

Abstract Nucleotide profiles of cell extracts were determined using high pressure liquid chromatography. The nucleotide levels were quantitatively reproducible and the adenine levels as determined by the analyzer were in close agreement with those determined by an enzymatic cycling procedure. Optimal conditions were determined for the mono-, di- and triphosphates of the naturally occurring ribosides as well as for the sulfur analogs of some of these compounds. The peaks were identified by the use of internal standards, by comparison to chromatograms of standard solutions, by collection and identification of the fractions chemically and spectrophotometrically and by the use of an enzymic peak-shift technique. The latter method which utilizes the specificity of enzyme reactions with a nucleotide or class of nucleotides, can be used not only to verify peak identities but also to clarify or “unmask” a chromatogram. Chromatograms of cell extracts of red blood cells, homogenized schistosomes or murine leukemia or sarcoma cells were obtained in seventy minutes.

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