A simple and rapid method for measuring α-D-phosphohexomutases activity by using anion-exchange chromatography coupled with an electrochemical detector.

The interconversion of hexose-6-phosphate and hexose-1-phosphate can be directly analyzed by high-performance anion-exchange chromatography coupled with an electrochemical detector (HPAEC-PAD). Thus, this method can be used to measure the activities of N-acetylglucosamine-phosphate mutase (AGM), glucosamine-phosphate mutase (GlmM) and phosphoglucomutase (PGM), which are the members of α-D-phosphohexomutases superfamily. The detection limits were extremely low as 2.747 pmol, 1.365 pmol, 0.512 pmol, 0.415 pmol, 1.486 pmol and 0.868 pmol for N-acetylglucosamine-1-phosphate (GlcNAc-1-P), N-acetylglucosamine-6-phosphate (GlcNAc-6-P), glucosamine-1-phosphate (GlcN-1-P), glucosamine-6-phosphate (GlcN-6-P), glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate (Glc-6-P), respectively. By employing HPAEC-PAD, activities of AtAGM (AGM from Arabidopsis thaliana) on these six phosphohexoses can be detected. The Km of AtAGM on Glc-1-P determined by HPAEC-PAD was 679.18 ± 156.40 µM, which is comparable with the Km of 707.09 ± 170.36 µM detected by traditional coupled assay. Moreover, the activity of MtGlmM (GlmM from Mycobacterium tuberculosis) on GlcN-6-P tested by HPAEC-PAD was 7493.40 ± 309.12 nmol∕min ⋅ mg, which is much higher than 288.97 ± 35.28 nmol∕min ⋅ mg obtained by the traditional coupled assay. Accordingly, HPAEC-PAD is a more rapid and simple method than the traditional coupled assays given its high specificity and sensitivity, and will certainly bring convenience to further research of α-D-phosphohexomutases.