High-pH anion-exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of nonpeptide mycobacterial antigens.

ABSTRACT

A rapid and sensitive high-pH anion-exchange chromatography (HPAEC) method for the separation and quantification of phosphorylated antigens in mycobacterial extracts has been developed. This method provides the separation of mono-, di-, or triphosphonucleotides and of various other phosphorylated molecules. Dual detection by conductimetry and UV absorption downstream of a chemical suppressor constitute nondegradative and highly sensitive tools for the physical detection and the quantification of phosphorylated compounds in biological samples. The lower limit of accurate quantification is around 1 nmol per sample. This method was used for the separation of several phosphorylated antigens activating human gamma delta T lymphocytes from semipurified mycobacterial fractions. Their quantification revealed that the minimal concentration activating a gamma delta T cell clone is between 1 and 5 nM. This approach can be used for more general preparative purposes with samples where minute amounts of biologically active phosphoanions are analyzed.