Camara Mohamed Amara, Tian Miaomiao, Guo Liping, Yang Li
Faculty of Chemistry, Northeast Normal University, Changchun, Jilin 130024, China.
Faculty of Chemistry, Northeast Normal University, Changchun, Jilin 130024, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 May 15;990:174-80. doi: 10.1016/j.jchromb.2015.03.019. Epub 2015 Apr 1.
In this study, we present an on-line measurement of enzyme activity and inhibition of Glucose-6-phosphate dehydrogenase (G6PDH) enzyme using capillary electrophoresis based immobilized enzyme micro-reactor (CE-based IMER). The IMER was prepared using a two-step protocol based on electrostatic assembly. The micro-reactor exhibited good stability and reproducibility for on-line assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme by six inhibitors, including three metals (Cu(2+), Pb(2+), Cd(2+)), vancomycin, urea and KMnO4, were investigated using on-line assay of the CE-based IMERs. The enzyme activity and inhibition kinetic constants were measured using the IMERs which were found to be consistent with those using traditional off-line enzyme assays. The kinetic mechanism of each inhibitor was also determined. The present study demonstrates the feasibility of using CE-based IMERs for rapid and efficient on-line assay of G6PDH, an important enzyme in the pentosephosphate pathway of human metabolism.
在本研究中，我们展示了一种基于毛细管电泳的固定化酶微反应器（CE-based IMER）用于在线测量葡萄糖-6-磷酸脱氢酶（G6PDH）的酶活性及抑制作用。该IMER采用基于静电组装的两步法制备。该微反应器在G6PDH酶的在线检测中表现出良好的稳定性和重现性。使用基于CE的IMER在线检测法研究了包括三种金属（Cu(2+)、Pb(2+)、Cd(2+)）、万古霉素、尿素和KMnO4在内的六种抑制剂对G6PDH酶的活性及抑制作用。使用IMER测量了酶活性和抑制动力学常数，发现其与传统离线酶分析法所得结果一致。还确定了每种抑制剂的动力学机制。本研究证明了使用基于CE的IMER对G6PDH进行快速高效在线检测的可行性，G6PDH是人类新陈代谢磷酸戊糖途径中的一种重要酶。
