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

Xiaochen Jia; Jian Kang; Heng Yin

doi:10.7717/peerj.1517

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

PeerJ. 2016 Jan 5:4:e1517. doi: 10.7717/peerj.1517. eCollection 2016.

Authors

Xiaochen Jia¹, Jian Kang², Heng Yin³

Affiliations

¹ Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; University of Chinese Academy of Sciences, Beijing, China.
² Department of Biochemistry and Molecular Biology, Dalian Medical University , Dalian , China.
³ Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian , China.

Abstract

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.

Keywords: HPAEC-PAD; N-acetylglucosamine-1-phosphate; N-acetylglucosamine-6-phosphate; N-acetylglucosamine-phosphate mutase; α-D-phosphohexomutases.

Grants and funding

This research was supported by the National Natural Science Foundation of China (31370811), the National Key Technology Support Program (2013BAB01B01) and the National High Technology Research and Development Program of China (2011AA10A205). Dr. Heng Yin was supported by the CAS Youth Innovation Promotion Association (2015144). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.