High pressure liquid chromatography of nucleotides and nucleic acid bases from fungi

High resolution liquid chromatographic methods have been developed for the determination of nucleotide pools at the nanogram level in four representative species of ascomycetes (P. citrinum, A. niger, F. moniliforme, and C. herbarum). Nucleotides were extracted from the mycelial mat in high yield with 10% (weight/volume) trichloroacetic acid, and then preseparated from interfering polysaccharides, glycoproteins, etc., on a Biegel P-2 column with double distilled water elution. Resolution of some 18 nucleotides from each fungal species is accomplished on AS-Pellionex-SAX pellicular anion exchanger using a high pressure liquid chromatograph. Identification of nucleotides was by comparing peak retention times; by differential ultra-violet absorption with two detectors in series at selected wavelengths; and by acid or enzymatic hydrolysis with product identification by liquid chromatography. Growth curves of the four fungi in liquid medium were monitored at 1-2 day intervals until growth stopped (~10 days). Pyrimidine bases were present in higher molar concentration than purines by a factor of at least three, with uridine nucleotides often representing 60- 80 mole percent of the total nucleotides. Extractable cytidine nucleotides are in negligible concentration in these species. Uridine diphosphoryl 2-acetamido-2-deoxy-α-D-glucopyranoside dominates all other nucleotides throughout the growth cycles of all four species, representing 30-60% of all nucleotides present.

A rapid quantitative procedure using high pressure liquid chromatography is developed for determining the percentage of guanine plus cytosine of DNA preparations after acid hydrolysis in 88% formic acid. As little as 10 μg DNA are amenable to this analysis. Although thymine is sometimes poorly resolved from impurities at the solvent front, percentage of G plus C was found to be accurately determined simply from the ratio of concentrations from C/(A+C) or (G+C)/(2A+G+C). The HPLC method gave values for the percentage G plus C in close agreement with the thermal melting point and buoyant density values determined for the DNA of ten bacterial species and two fungi isolated for this study.