A simple and rapid method for measuring α-D-phosphohexomutases activity by using anion-exchange chromatography coupled with an electrochemical detector.
A simple and rapid method for measuring α-D-phosphohexomutases activity by using anion-exchange chromatography coupled with an electrochemical detector.
复制标题
一种使用阴离子交换色谱与电化学检测器联用测量α-D-磷酸己糖变位酶活性的简单快速方法
作者：
Jia X;Kang J;Yin H
己糖 - 6 - 磷酸和己糖 - 1 - 磷酸的相互转化可通过高效阴离子交换色谱与电化学检测器联用（HPAEC - PAD）直接进行分析。因此，该方法可用于测量N - 乙酰葡糖胺 - 磷酸变位酶（AGM）、葡糖胺 - 磷酸变位酶（GlmM）和磷酸葡萄糖变位酶（PGM）的活性，它们是α - D - 磷酸己糖变位酶超家族的成员。N - 乙酰葡糖胺 - 1 - 磷酸（GlcNAc - 1 - P）、N - 乙酰葡糖胺 - 6 - 磷酸（GlcNAc - 6 - P）、葡糖胺 - 1 - 磷酸（GlcN - 1 - P）、葡糖胺 - 6 - 磷酸（GlcN - 6 - P）、葡萄糖 - 1 - 磷酸（Glc - 1 - P）和葡萄糖 - 6 - 磷酸（Glc - 6 - P）的检测限极低，分别为2.747皮摩尔、1.365皮摩尔、0.512皮摩尔、0.415皮摩尔、1.486皮摩尔和0.868皮摩尔。通过采用HPAEC - PAD，可以检测拟南芥AGM（AtAGM）对这六种磷酸己糖的活性。通过HPAEC - PAD测定的AtAGM对Glc - 1 - P的Km值为679.18 ± 156.40微摩尔，与传统偶联测定法检测到的707.09 ± 170.36微摩尔的Km值相当。此外，通过HPAEC - PAD检测的结核分枝杆菌GlmM（MtGlmM）对GlcN - 6 - P的活性为7493.40 ± 309.12纳摩尔/分钟·毫克，远高于传统偶联测定法得到的288.97 ± 35.28纳摩尔/分钟·毫克。因此，鉴于HPAEC - PAD具有高特异性和高灵敏度，它是一种比传统偶联测定法更快速、更简便的方法，必将为α - D - 磷酸己糖变位酶的进一步研究带来便利。
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.