Pyrimidine nucleotide chromatography

In view of the difficulty of hydrolyzing the pyrimidine nucleosidic linkages, ribonucleic acids have been hydrolyzed to a mixture of purine bases and pyrimidine nucleotides which is then separated by paper chromatography.132, 163 164 This method has been employed extensively for the analysis of ribonucleic acids, and gives reproducible results,166 but it has not been used to any great extent for deoxyribonucleic acids, probably because, under these conditions of hydrolysis, they yield some pyrimidine deoxy-ribonucleoside diphosphates.166... 

The separation of nucleotides and deoxynucleotides, previously a formidable task involving the fractional crystallization of heavy metal and alkaloid salts 102) has been made much easier by developments in analytical techniques. Ion-exchange methods may be used for the purification, isolation, and identification of both classes of nucleotides from hydrolysis mixtures 103), Countercurrent distribution 104) and starch 106) and cellulose-column 106) as well as paper-strip chromatography 107) have also proved to be useful in separating nucleotides from natural sources. Spectro-photometric procedures based on the characteristic ultraviolet absorption spectra of the purines and pyrimidines have been the most convenient method to locate, estimate, and identify the fractions obtained in the previous separations. Since the nucleotides are acid in nature, they are often named as acids, e.g., adenylic acid, cytidylic acid. The general constitution of the purine nucleotides (and by analogy the pyrimidine nucleotides) is demonstrated by their hydrolysis by acids to a purine and ribose (or 2-deoxyribose) monophosphate and by alkalies to the nucleosides and phosphoric acid. The order of the constituents in a purine nucleotide must, therefore, be ... 

Fig. 2.1. Nucleotide fractionation. A sample of p2 phage RNA uniformly labelled with and the pyrimidines labelled with was digested with alkali ( 2.1.1) and separated by column chromatography on Dowex 50 formate eluted with 0.25 M ammonium formate pH 4.1. The ultraviolet transmission graph is traced from the record of a Uvicord I ultraviolet monitor output Radioactivities were measured by dissolving the samples in a scintillation cocktail and counting in a liquid scintillation counter. The peaks were identified from their ultraviolet spectra.

See also Electrophoresis Two-Dimensional Gels Nucleic Acids. Enzymes Enzyme-Based Assays. Flow Injection Analysis Principles. Fluorescence Quantitative Analysis. Lab-on-a-Chip Technologies. Mass Spectrometry Matrix-Assisted Laser Desorption/loniza-tion Time-of-Flight. Microelectrodes. Microscopy Overview. pH. Process Analysis Overview Chromatography Electroanalytical Techniques Sensors Acoustic Emission Maintenance, Reliability, and Training. Proteins Overview. Proteomics. Purines, Pyrimidines, and Nucleotides. Sensors Oven/iew. Spectrophotometry Overview. 

Liquid chromatography is a rapid, selective and sensitive technique which can be adapted to the analysis of extracts from a variety of biological sources. It constitutes an excellent method for the analysis of nucleotides, nucleosides, purines, and pyrimidines bases. 

From Fig. 1 (a,b,c, and d) it is clear that good separation of each ribonucleotide and deoxynucleotide in all purine and pyrimidine series is achieved on a C g column, using isocratic elution with ammonium phosphate buffer (0.2M, pH 5.1). Fig. 2 (a,b,c, and d) illustrates the effect of periodate treatment on the ribo-nucleotide-deoxynucleotide mixture in each series. In the chromatography of ribonucleotides, the triphosphate always is eluted first, closely followed by the diphosphate, with the monophosphate being retained for a relatively longer period. A similar pattern emerges with the deoxynucleotides. After periodate treatment, we have noted a minor shift in the elution pattern. 

High pressure liquid chromatography has proved to be a valuable tool in measuring the nucleotide levels in cell extracts (1, 2). The analyses are accomplished with high speed, sensitivity, resolution and accuracy. A decided advantage of this technique is that the major naturally occurring nucleotides can be monitored at one time concurrently with nucleotides of purine and pyrimidine analogs. 

---