Quantitation of guanosine 5',3'-bisdiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography

A new method to determine the guanosine 5′,3′-bisdiphosphate (ppGpp) concentration in bacterial cultures has been developed. Cells in which the nucleotide pool was stabilized by treatment with formaldehyde were concentrated from samples of cultures and lysed by alkali. The ppGpp was separated from other nucleotides on a reverse phase C₁₈ column by ion-pair high performance liquid chromatography using an isocratic elution system. The area of the peak corresponding to ppGpp in elution profiles obtained by monitoring the absorbance of the eluant at 254 nm was used to quantitate ppGpp. The method allows detection of as little as 1 pmol of ppGpp per A₄₆₀ of culture, with $\text{2.5}\text{pmol}\text{A}{}_{460}$ being quantitated with greater than 90% accuracy.