31P NMR Examination of Phosphorus Metabolites in the Aqueous, Acidic, and Organic Extracts of Phaseolus vulgaris Seeds

Abstract

31P NMR spectroscopy was used to compare the phosphorus compound content in aqueous, acidic, and organic extracts of Phaseolus vulgaris seeds. Two chloroform-methanol and three ethanol extractions were used to isolate phospholipids from dry cotyledons and the phospholipid profiles were compared. Variations in phospholipid composition and artificially high concentrations of lysophosphatidylcholine were observed in the ethanol extracts. Other phosphorus compounds were extracted using perchloric acid, trichloroacetic acid, trichloroacetic acid in ether, hydrochloric acid, boiling water, and aqueous Hepes. The Hepes extraction was introduced in order to compare the results of a gentle procedure with those involving heat and acid treatments. Low concentrations of phosphosugars and other phosphorus metabolites were found in all these extracts. High concentrations of phytate were found in all aqueous and acidic extractions; however, 31P NMR spectra of aqueous extractions did not show the phytate resonance. The NMR silence of the phytate resonances is attributed to complexation of phytate. The 31P NMR spectra of aqueous extracts contained a broad resonance at 0.1 ppm assigned to protein-bound RNA. Various extraction procedures on the same biological material are presented, and the comparison of these methods will facilitate future analysis and interpretation of literature reports involving these methods of tissue analysis.